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Breeding and propagation of Meconopsis Qu, Yong 1985

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BREEDING AND PROPAGATION OF MECONOPSIS By YONG QU B.Sc, Beijing Forestry University, 1982 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Department of Plant Science) We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA September 1985 © Yong Qu, 1985 In presenting t h i s thesis i n p a r t i a l f u l f i l m e n t of the requirements for an advanced degree at the University of B r i t i s h Columbia, I agree that the Library s h a l l make i t f r e e l y available for reference and study. I further agree that permission for extensive copying of t h i s thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. I t i s understood that copying or publication of t h i s thesis for f i n a n c i a l gain s h a l l not be allowed without my written permission. Department of The University of B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date j^Jjtespnbetf ^4 19 ABSTRACT Six species of Meconopsis were investigated for self-f e r t i l i t y and cross compatibility in order to incorporate desirable characters from both parents. The six species used were: M_;_ cambrica, M. vill o s a , M. quintuplinervia, M.  betonicifolia, M. horridula, M. aculeata. Chromosome counts were made: M. cambrica, n=14; M. vi l l o s a , n=16; M. quintuplinervia, n= c.42; M. betonicifolia, n=40; M. horridula, n=28 and M. aculeata, n=28. Interspecific compatibility was correlated with differences in ploidy level between parents. A l l crosses made except M.  cambrica x quintuplinervia set seeds. cambrica, M. betonicif o l i a , M. horridula and M_;_ aculeata are self-compatible. Pollination mechanisms and the likelihood of apogamy were investigated in VL_ betonicif o l i a . No apogamy was found and insects are the likely pollinators for this species. As some of the species do not flower at the same time, pollen staining, pollen germination and storage conditions for the six species were studied. Experimental alteration of flowering periods through controlling temperatures and day-lengths for betonicifolia was also carried out. This part of i i the project shows: (1) pollen stainability (stained by lactophe-nol cotton blue) for the six species was 85% or more; (2) pollen of a l l six species except VL_ quintuplinervia germinated on an agar medium containing sucrose (5 g l--*-) and H3BO3 (0.1 mg I--*-); (3) pollen germination percentage decreased with storage time in a desiccator at 4-6°C; and (4) long daylength (16 h) was suitable for growth and flowering of betonicifolia. High temperature (17°C) induced earlier growth and flowering than low temperature (6°C) in M^_ betonicifolia. Because of d i f f i c u l t i e s in vegetative reproduction and seed storage, _in_ vitro establishment of M_;_ betonicif o l i a was investigated. The unopened capsule st e r i l i z a t i o n method was used. The seeds in the capsule germinated on Murashige and Skoog medium. Derooted seedlings, hypocotyls and seedling roots from seedlings raised on sterile a r t i f i c i a l medium were used as explants for the in vitro establishment. This experiment shows that half strength Murashige and Skoog medium is suitable for in vitro culture of this species. The cytokinin:auxin ratio and growth regulator concentration were found to control morphogenesis of M_;_ betonicif o l i a in vitro. Derooted seedlings cultured f i r s t on nutrient medium containing -benzyladenine i i i (BA) and kine'tin (Kn) differentiated more multiple meristems than those originally cultured on medium containing N^-(2-isopentenyl)-adenine (2ip) after being transferred into medium containing 2ip. Multiple meristems were divided and proliferated on medium containing 0.2 mg 1~1 2,4-dichlorophenoxyacetic acid (2,4-D), 5 mg 1_1 2ip and 1 mg 1-1 BA. Seedling roots and hypocotyls formed callus on media containing 1, 5 and 10 mg 1-1 2,4-D. iv ABBREVIATIONS MS Murashige and Skoog medium (1962) IAA Indole-3-acetic acid NAA 1-Naphthaleneacetic acid 2,4-D 2,4-Dichlorophenoxyacetic acid BA N^-Benzyladenine Kn Kinetin 2ip N6-(2-isopentenyl)-adenine LD Long day-length SD Short day-length HT High temperature LT Low temperature v TABLE OF CONTENTS Contents page ABSTRACT i i ABBREVIATIONS v TABLE OF CONTENTS vi LIST OF TABLES v i i i LIST OF FIGURES x ACKNOWLEDGEMENT x i i i GENERAL INTRODUCTION 1 SECTION A. S e l f - f e r t i l i t y and cross compatibility among six species of Meconopsis 9 Introduction 9 Materials and methods 14 Results 17 Discussion 27 Conclusions 33 SECTION B. Pollen staining and germination test for six species in Meconopsis 34 Introduction 34 Materials and methods 36 vi Results 38 Discussion 45 Conclusions 51 SECTION C. Influence of temperature and day-length on flowering of Meconopsis betonicifolia 52 Introduction 52 Materials and methods 54 Results 55 Discussion 57 Conclusions 66 SECTION D. In vitro establishment of Meconopsis betonicifolia 67 Introduction 67 Materials and methods 72 Results 82 Discussion 97 Conclusions 107 OVERALL DISCUSSION 109 OVERALL CONCLUSIONS AND SUMMARY 114 BIBLIOGRAPHY 118 v i i LIST OF TABLES Table page 1. Selfing and crossing among six species of Meconopsis 18 2. Pollination treatments for Meconopsis betonicifolia 26 3. Analysis of compatibility in Meconopsis based on chromosome ploidy level 28 4. Pollen staining percentage in six species of Meconopsis 40 5. Pollen germination percentage in six species of Meconopsis 43 6. Daylength and temperature change in relation to growth and flowering of Meconopsis betonicifolia 56 7. Survival percentage of derooted seedlings of Meconopsis betonicifolia after treatment with 2,4-D , cytokinin and strength of MS medium 84 8. Analysis of variance of Table 7 85 9. Summary of test results from Tables 7 and 8 86 10. Differentiation percentage with different concentrations of BA, Kn or 2ip 87 11. Analysis of variance of Table 10 88 v i i i 12. Differentiation percentage with different 2ip and 2,4-D concentration after subculture from BA, Kn or 2ip media 89 13. Analysis of variance of Table 12 90 14. Summary of test results from Tables 12 and 13 91 ix LIST OF FIGURES Figures page 1. Method of selfing and crossing 15 2. Relationship between percentage of capsules setting seeds and seeds per pollination in Meconopsis 19 3. Relationship between percentage of capsules setting seeds and seeds per capsule in Meconopsis 20 4. Relationship between seeds per pollination and seeds per capsule in Meconopsis 21 5. Meiotic chromosomes of Meconopsis cambrica 22 6. Meiotic chromosomes of Meconopsis betonicifolia 23 7. Rank il l u s t r a t i o n of s e l f - f e r t i l i t y and cross compatibility based on seeds per pollination 24 8. Stained pollen grains of Meconopsis aculeata 41 9. Stained pollen grains of Meconopsis betonicifolia 42 10. Pollen tube growth of Meconopsis aculeata 44 11. Fresh pollen germination of Meconopsis betonicifolia on 0.5% agar medium 46 x 12. Fresh pollen of Meconopsis betonicifolia on 1% agar medium 47 13. Flowering of Meconopsis betonicifolia at low temperature (6°C) 58 14. Flowering of Meconopsis betonicifolia at high temperature (17°C) 59 15. Potted seedlings of Meconopsis betonicifolia after 70-day short day treatment at 6°C 62 16. Potted seedlings of Meconopsis betonicifolia after 70-day long day treatment at 6°C 63 17. In vitro shoots from derooted seedlings of Meconopsis betonicifolia 75 18. Iri vitro shoots from derooted seedlings of Meconopsis betonicifolia (showing callus at base of shoots) 76 19. Multiple meristems differentiated from derooted seedlings of Meconopsis betonicifolia 77 20. Multiple meristems differentiated from callus induced from derooted seedlings of Meconopsis  betonicifolia 78 21. Multiple shoots differentiated from derooted xi seedlings of Meconopsis betonicifolia 79 22. Responses of derooted seedling and seedling root cultures from light-raised seedlings to 2ip and 2,4-D concentrations and the ratio of 2ip to 2,4-D by weight 93 23. Responses of hypocotyl and seedling root cultures from dark-raised seedlings to 2ip and 2,4-D concentration and the ratio of 2ip to 2,4-D by weight 94 24. Callus induced from hypocotyls of Meconopsis betonicifolia 95 25. Callus induced from seedling roots of Meconopsis betonicifolia 96 x i i ACKNOWLEDGEMENTS I wish to express ray appreciation for assistance in this study to: My committee members: Dr. Colin R. Norton and Dr. Victor C. Runeckl-es (Department of Plant Science), Dr. Gerald B. Straley (Botanical Garden Office) and Dr. Fred R. Ganders (Department of Botany), who provided guidence, advice and criticisms of the thesis. Dr. George W. Eaton for guiding me in s t a t i s t i c a l analysis of some data and Dr. Margaret E. Norton for allowing me to read her Ph.D thesis about tissue culture. Staff of Botanical Garden and Nursery, and VanDusen Botanical Garden for information and materials. Technicians in the Department of Plant Science for their assistance in repairing equipment, timely provision of materials and some photographs. I would also like to thank the Chinese Government for my scholarship and my friends in China and Canada for their encouragement. x i i i General Introduction The genus Meconopsis, which belongs to the family Papaveraceae, was named by Viguier in 1814. It was based on the single species Papaver cambricum, and is differentiated from Papaver by the presence of a short style and the complete absence of a sessile stigmatic disc surmounting the ovary (Taylor, 1934). Meconopsis consists of 49 species. One of them is native to western Europe whilst the others are a l l distributed in the Sino-Himalayan Region of Asia (Wu and Chuang, 1980). With the exception of M_;_ cambrica, which inhabits damp, shady ravines at altitudes usually below 600 m, Meconopsis occurs in alpine woods, scrub, meadows, and scree slopes above 1,800 m. Several species grow at elevations above 5,000 m (Taylor, 1934). 'The genus, like other alpine plants, produces comparatively large, b r i l l i a n t l y coloured flowers. Flower colours are blue, yellow, white, red and purple. This genus is horticulturally useful (Taylor, 1934). The taxonomy (Prain, 1906; Fedde, 1909; Taylor, 1934; Wu and Chuang, 1980), cytology (Maude, 1940; Ernst, 1965; Ratter, 1968), 1 pollen morphology (Henderson, 1965), hybrids (Taylor, 1934, 1951; Evans, 1959; Ramsden, 1962; Cobb, 1984), seed germination (Amsler, 1945; Thompson, 1968, 1969; Anonymous, 1984), cultivation and horticultural value (Harley, 1928; Cox, 1934; Harrow, 1930; Evans, 1974) of Meconopsis have been studied, but there are s t i l l some unresolved problems. Most members of Meconopsis are monocarpic. Only a few species are polycarpic (Taylor, 1934; Evans, 1959). Within the genus, several controlled interspecific crosses have been reported between polycarpic species and monocarpic species (Taylor, 1934; Evans, 1959). The hybrids closely resembled their polycarpic parents. This shows that the possibility of controlled crossing between species exists. This fact stimulated interest in incorporating desirable f l o r a l habit from monocarpic species into polycarpic species to increase longevity of plants with desirable flower colour, number and size. The f e a s i b i l i t y of such a breeding program is unknown because l i t t l e i s known about self -compatibility, interspecific cross compatibility, the pollination mechanisms or the possibility of apogamy in the genus. Thus, a hybridization and selfing test was designed to investigate self and cross pollination among the six species of this genus grown 2 in Vancouver. The hypotheses of this experiment were: (1) interspecific crosses are compatible among the six species; and (2) the species in this genus are self-compatible. If these hypotheses are true, i t i s possible to get hybrid seeds and seeds from selfed plants. The six species available for study were: cambrica (L.) Vig., quintuplinervia Regel, M. betonicifolia Franch., M. horridula Hook f. & Thorns, and M_;_ aculeata Royle (grown in the University of British Columbia Botanical Garden), and M. villosa (Hook, f.) G. Tayl. (grown in the VanDusen Botanical Garden, Vancouver). Possible pollination mechanisms and the possibility of apogamy were investigated in betonicifolia. M. betonicifolia was used because of i t s high horticultural usefulness, i t s pure blue flowers and availability of adequate plant material. The morphological differences among these six species are: M. cambrica: polycarpic, yellow to orange flower, tufted base of plant glabrous or sparsely pubescent, lamina of basal leaves pinnatisect towards the base and pinnatifid towards the apex. M. vi l l o s a : polycarpic, yellow flower, tufted base of plant 3 beset with barbellate rufous h a i r s , lamina of basal leaves ovate to suborbicular, deeply bipinnately lobed. M. q u i n t u p l i n e r v i a : polycarpic, flower pale lavender-blue, leaves a l l basal, pendulous flowers borne on basal scapes. M. b e t o n i c i f o l i a : monocarpic or polycarpic, flower colour ranging from rose-lavender to satiny-sky-blue, leaves basal and cauline. M. h o r r i d u l a : monocarpic, flower l i g h t - b l u e to c l a r e t , l e a f -lamina e n t i r e or r a r e l y pinnately lobed at the margin. M. aculeata: monocarpic, flower purplish-blue, leaf-lamina deeply p i n n a t i f i d or p i n n a t i s e c t . Among the s i x species investigated, M_;_ q u i n t u p l i n e r v i a flowers e a r l i e s t ( i n A p r i l ) while VL_ v i l l o s a flowers i n June i n Vancouver. Their blooming times do not overlap. Consequently, an i n t e r s p e c i f i c breeding program involving these species would require pollen storage or experimental a l t e r a t i o n of flowering periods. Thus a pollen v i a b i l i t y t e s t was designed to investigate pollen s t a i n i n g , pollen germination and storage conditions for 4 the six species. The experiment to alter flowering time was designed to test the hypothesis that different temperatures and day-lengths can change flowering time of the species in Meconopsis in growth chambers. No such information was available from earlier studies. Such data would be helpful in improving breeding techniques for this genus by allowing controlled pollination between species that do not flower at the same period. Meconopsis i s usually raised from seeds. Vegetative reproduction i s possible in only a few perennial species (Cox, 1934). Most Meconopsis are monocarpic so that regular successful seed set each year i s essential for their cultivation. Under normal storage conditions the seed remains viable over only relatively short periods of a year or two so that continued cultivation of individual species i s dependent f i r s t on their capacity to set seed under garden conditions, and then on the abi l i t y of the gardener to raise seedlings and bring them into flower. Adverse circumstances may occasionally prevent the raising of young plants and even a single generation lost in this way may lead to the loss of an entire stock. Very many attractive species which have flowered from seeds collected in the wild 5 have, after a few years, been lost to cultivation because of failures to maintain successful stocks of seedlings (Thompson, 1968). Hence, i t is desirable to develop an alternative propagation method to ensure continued success in cultivation of particularly attractive clones. Cox (1934) pointed out that summer heat, particularly when humidity i s low, is the main reason for failure of Meconopsis in the south of England, in eastern and central Canada and the United States. Cultivars which could resist summer heat and low humidity would extend the range of cultivation of this genus. Following the successful rapid multiplication of orchids by shoot meristem culture (Morel, 1965) there has been an increasing interest in recent years in the application of tissue culture techniques as an alternative means of asexual propagation of economically important plants. Potential advantages of the in  vitro micropropagation are: (1) recovery of pathogen-free stock (Morel, 1960); (2) providing increased rates of propagation and fa c i l i t a t i n g asexual multiplication of plants that had previously proved d i f f i c u l t , or impossible, to propagate (Hussey, 1978); (3) potential benefits in long-term storage (Morel, 1975) and cryogenic preservation of germplasm (Seibert, 1976); (4) 6 international transport of genetic stocks (Kahn, 1976); and (5) proliferating the in vitro stocks at any time of the year (Boxus et a l . , 1977) after shipping or storage, while propagation with conventional practices i s highly season-dependent. From the breeder's point of view, the tissue culture technique provides an effective tool to release new varieties rapidly (Hu et a l , 1982). The production of haploid plants, specific desirable gene combination and regenerates with desirable characters from somaclonal variation can be very useful in plant breeding. If a successful method of tissue culture could be worked ' out for Meconopsis, i t may help not only propagation, but also preservation of stock, and breeding of this genus. No information about tissue culture of Meconopsis was available. Meconopsis betonicifolia was chosen to conduct the in  vitro culture experiment because of i t s horticultural usefulness and plant material a v a i l a b i l i t y . The object of this, experiment was to determine an appropriate explant and an optimal medium for i t s growth and multiplication in vitro. The ultimate objectives of the project were: (1) investigation of some plant characters related to breeding of Meconopsis; and (2) development of a new propagation method for 7 M. betonicifolia. The -thesis shows methods for pollen staining, pollen storage and germination testing, the f e a s i b i l i t y of hybridization and selfing, and the relationship between interspecific compatibility and chromosome ploidy level of six species in this genus. Data regarding possible pollination mechanisms, apogamy, factors controlling flowering time, and the in vitro establishment of M. betonicifolia are also given. 8 Section A. S e l f - f e r t i l i t y and Cross Compatibility among Six Species of Meconopsis Introduction Within the genus Meconopsis, some hybrids were reported from previous studies. Taylor (1934) pointed out that with few exceptions the hybrids have arisen spontaneously and evidence of their parentage i s therefore often hypothetical. The reported hybrids were: (1) Meconopsis x Harleyana: M. in t e g r i f o l i a x M.  simplicifolia (Taylor, 1934); (2) M. x Auriculata: M. betonicifolia x M^_ paniculata (Taylor, 1934); (3) M^ x Coxiana: M. betonicifolia x M_;_ violacea (Taylor, 1934); (4) x Musgravei: M. betonicifolia x M_^_ superba (Taylor, 1934); (5) Hj_ x Sarsonsii: M. betonicifolia x M_;_ integrif o l i a (Taylor, 1934); 9 (6) Hj_ x Beamishii: M. grandis x integr i f o l i a (Taylor, 1934); (7) x Hybrida: M. grandis x simplicifolia (Taylor, 1934); (8) M_;_ x Decora: M. l a t i f o l i a x napaulensis (Taylor, 1934); (9) Hj_ x Cookei: M. punicea x quintuplinervia (Taylor, 1951); (10) x Finlayorum: M. int e g r i f o l i a x quintuplinervia (Taylor, 1951); (11) M_;_ x Sheldonii: M. grandis x betonicif o l i a (Evans, 1959); (12) ML_ x Ramsdeniorum: M. dhwojii x napaulensis (Ramsden, 1962); (13) Unnamed: M. quintuplinervia x M. cambrica (Cobb, 1984); (14) Unnamed: M. simplicifolia x M. paniculata (Cobb, 1984); (15) Unnamed: M. grandis x M. regia (Cobb, 1984). Of the fifteen hybrids, five were controlled crosses. Two hybrids from controlled crosses: x Musgravei (M. betonicifolia x M_j_ superba) and x Sarsonsii (M. betonicif o l i a x 10 integrifolia) were described as closely resembling the polycarpic parent (M. betonicifolia) and showing the flower colour influence of monocarpic parents (M. superba and integrifolia) (Taylor, 1934). This indicates the possibility of controlled crossing between species within the genus. Thus i t i s possible to incorporate desirable characters from both parents to create new varieties. The fe a s i b i l i t y of self-compatibility, cross compatibility, pollination mechanisms or possibility of apogamy in the genus i s unknown. The object of this experimental pollination study was to investigate self-compatibility and interspecific cross compatibility among the six species in this genus, which are cultivated in the University of British Columbia Botanical Garden and VanDusen Gardens, Vancouver. No controlled crossing and selfing among the six species were reported from previous studies except one unnamed hybrid: M.  quintuplinervia x M. cambrica (Cobb, 1984). Meconopsis betonicifolia was chosen to study pollination mechanisms and the likelihood of apogamy for two reasons: (1) horticultural usefulness of i t s pure blue flower; and (2) plant material availablity. 11 Chromosome numbers have been determined for eighteen species in the genus. They vary from n=14 to n=60 within the genus (Ratter, 1968). Chromosome numbers reported for the six species were: M. cambrica, n=ll (Maude, 1940), n=14 (Sugiura, 1937, 1940; Ratter, 1968), n=28 (Ernst, 1962, 1965); M. vi l l o s a , n=16 (Ernst, 1962, 1965; Ratter, 1968); M^_ quintuplinervia, n=42 (Ratter, 1968); M. betonicifolia, n=41 (Ratter, 1968), n=40 (Ernst, 1965); M. horridula, n=28 (Ratter, 1968); and M. aculeata, n=28 (Ratter, 1968). Thus, this study was also designed to determine chromosome number for the six species cultivated in the University of British Columbia Botanical Garden and VanDusen Garden. Previous studies about chromosome number for the six species show that they have different ploidy levels (Ratter, 1968). If this i s true for the six species grown in Vancouver, then interspecific crosses can be tested between species with the same or different ploidy levels. The degree of interspecific cross compatibility can be compared with difference in ploidy level between parents. The hypotheses of this experiment were: (1) the degree of compatibility between species differs with difference in ploidy level between parents; 12 species of this genus are self-compatible M. betonicifolia i s not apogamous. 13 Materials and methods Plants of Meconopsis cambrica, M. vi l l o s a , M.  quintuplinervia, M. betonicifolia, M. horridula and M^ aculeata cultivated in the two Botanical Gardens were used for selfing and crossing experiments. Three to eighteen replicates were used depending on plant av a i l a b i l i t y . The availability of plant materials and difference in flowering time prevented a l l possible crosses from being made. For each selfing and crossing, the stamens were removed using a pair of tweezers just before corolla opening. At this stage, anthers had not dehisced but stigmas were receptive. Fresh pollen was applied to the stigma of the female parent. Pollen of M. quintuplinervia stored in a refrigerator at 4-6°C was used because of differences in flowering time. The pollinated flower was covered with a waxed paper bag (Figure 1) and the bag was removed when the stigma withered. Capsules were collected when mature and open. Seeds from capsules were examined under a dissecting microscope. Seeds which looked flattened and non-viable were not counted while those which looked plump were recorded for each crossing and selfing. Where very large numbers 14 15 of seeds were set, they were recorded as greater than 300. Seeds from a l l successful crosses were sown in the pots by staff of the Botanical Garden Nursery at the University of British Columbia in the spring of 1985. Pollination treatments used in betonicifolia were: (1) exposure to natural environment after emasculation; (2) bagging flowers without emasculation; (3) bagging flowers after emasculation; (4) pollination using different plants of the same species; and (5) pollination using the same flower. For chromosome counts, whole flower buds were fixed in ethanol:chloroform:acetic acid (6:3:1) after pretreatment with p-dichlorobenzene. They were stored in a refrigerator and squashed in acetocarmine (4 g in 100 ml 45% acetic acid 1 _1). The slides were observed using a Photomicroscope II (Carl Zeiss Oberkochen/West Germany). In the following text, the combination: "A species x B species" means A as female and B as male parents; the combination: "A species x A species" means selfing. 16 Results Percentage of capsules setting seeds (fruit set), seeds per pollination and seeds per capsule were calculated as measures of selfing and cross compatibility (Table 1). They were a l l linearly correlated: Seeds per pollination and f r u i t set, r=0.74, (p<0.01, Figure 2); seeds per capsule and frui t set, r=0.58 (p<0.05, Figure 3); seeds per capsule and seeds per pollination, r=0.90 (p<0.01, Figure 4). The meiotic chromosome numbers found were: M. cambrica, n=14 (Figure 5) and NL_ vill o s a , n=16 (these represent tetraploidy with x=7 and x=8 respectively); M. quintuplinervia, n (approximate)=42 and betonicifolia, n=40 (Figure 6) (these are nearly duodecaploids); and horridula, n=28, and M. aculeata, n=28 (octoploids). Since the number of seeds per pollination i s highly correlated with both f r u i t set and seeds per capsule, i t was used as the index to represent the degree of compatibility. Figure 7 illustrates a l l selfing and crossing results in Table 1. It can be seen clearly that: (1) of those species selfed, compatibility was highest in M_;_ cambrica, M. betonicif o l i a and horridula, 17 Table 1. Selfing and crossing among six species of Meconopsis cross rank no. of no. of % of total no. of no. of (F ig.7) flowers capsules capsules seed seeds per seeds pollinated setting setting number p o l l i - per seeds seeds nation capsule (1) (2) (3) (4) (5) (6) (7) (8) M.c.x M.c. 1 11 9 82 >300 >27.3 >33.3 M.c.x M.v. 1 15 5 33 128 8.5 25.6 M.c.x M.q. 3 14 0 0 0 0 0 M.c.x M.b. 3 11 2 18 6 0.6 3.0 M.c.x M.h. 2 16 4 25 28 1.8 7.0 M.c.x M.a. 3 15 3 20 7 0.5 2.3 M.v.x M.a. 1 4 4 100 >300 >75.0 >75.0 M.b.x M.c. 3 15 3 20 6 0.4 2.0 M.b.x M.v. 2 15 9 60 70 4.7 7.8 M.b.x M.q. 1 15 5 33 98 6.5 19.6 M.b.x M.b. 1 18 8 100 >300 >16.7 >16.7 M.b.x M.h. 2 15 6 40 42 2.8 7.0 M.b.x M.a. 1 12 10 83 180 15.0 18.0 M.h.x M.c. 2 6 3 50 15 2.5 5.0 M.h.x M.b. 3 3 1 33 2 0.7 2.0 M.h.x M.h. 1 15 3 20 135 9.0 45.0 M.h.x M.a. 1 10 8 80 >300 >30.0 >37.5 M.a.x M.a. 2 10 3 30 18 1.8 6.0 M.c.= M. cambrica; M.v.= M. villo s a ; M.q.= M. quintuplinervia M.b.= M. betonicifolia; M.h.= M. horridula; M.a.= M. aculeata 18 2. Relationship between percentage of capsules s e t t i n g seed and seeds per p o l l i n a t i o n i n Meconopsis (from Table column 5 and 7). 19 80 i 70 y = 0 . 2 6 4 3 + 0 .3725x r = 0 . 5 7 9 9 ( p < 0 . 0 5 ) • t —r -0 10 20 30 40 50 60 70 80 90 100 p e r c e n t a g e o f c a p s u l e s s e t t i n g seeds . 3. Relationship between percentage of capsules setting seeds and seeds per capsule in Meconopsis (from Table 1 columns 5 and 8). 20 0 ^ 1 1 1 ! r— 0 10 20 30 40 50 60 70 80 90 100 number o f seeds per p o l l i n a t i o n Fig. 4. Relationship between seeds per pollination and seeds per capsule in Meconopsis (from Table 1 columns 7 and 8). 21 Fig. 5. Meiotic chromosomes of Meconopsis cambrica (xlOOO). 22 Fig. 6. Meiotic chromosomes of Meconopsis betonicifolia (x400). 23 n=approximate 42 n=40 n=14 male H female Fig. 7. Rank il l u s t r a t i o n of s e l f - f e r t i l i t y and cross compatibility based on seeds per pollination (Species which are on the same horizontal line are at the same ploidy level). Legend: sign compatibility Rank seeds per pollination 1 > 5 2 1.8—5 3 < 1.8 highest lowest Q=M. quintuplinervia; B=M. betonicifolia; A=M. aculeata; H=M. horridula; V=M. vi l l o s a ; C=M. cambrica. 24 and lowest in aculeata; and (2) the interspecific cross compatibility was highest between species at the same ploidy level and between M_;_ betonicifolia x M^ aculeata and M_^_ v i l l o s a x M. aculeata. Seeds set from these crosses exceeded 5 seeds per pollination. Other crosses gave low seed set (less than 5 seeds per pollination). Seeds sown in the Botanical Garden Nursery a l l germinated and grew into seedlings. The f i r s t hybrid (M. cambrica x M.  horridula) flowered on 29 May, 1985. This hybrid showed the intermediate characters of both parents. For example, flower colour was purple at the base of the petals (M. horridula has purple flowers) and was orange at the top of the petals (M.  cambrica has yellow to orange flowers)(information about seed germination and the hybrid produced were from the Botanical Garden Nursery of the University of British Columbia). Pollination treatments in M^_ betonicifolia (Table 2) showed that: (1) exposure to the natural environment, after emasculation, gave 93% capsule set. (2) 18% of capsules set seeds after bagging flowers. (3) No seeds set when flowers were bagged after emasculation. (4) Selfing and intraspecific crossing gave 100% f r u i t set (treatments IV and V). 25 Table 2. Pollination treatments for Meconopsis betonicifolia no. of no. of total % of seeds seeds no treatment flowers capsules seeds capsules per per pollinated setting number setting p o l l i - capsule seeds seeds nation I. emasculation + 15 14 >300 93 >20.0 >21.4 natural exposure II. bagging flowers 11 2 17 18 1.6 8.5 III. emasculation + 10 0 0 0 0 0 bagging IV. pollen from 9 9 >300 100 >33.3 >33.3 different plant V. pollen from the 11 11 >300 100 >27.3 >27.3 same flower 26 Discussion According to difference in ploidy level between parents, selfing and crossing carried out in the current experiment were divided into four groups (Table 3). In Groups I, II and III, the differences in chromosome ploidy level between parents are 8, A and 0 respectively. Group IV shows the selfing results. The correlation analysis showed that the greater the difference in ploidy level between parents, the less compatibility between species. The correlation between ploidy level differences and compatibility ranks was highly significant with r=0.74 (p<0.01). The fact that compatibility (measured by seed set ability) was correlated with differences between parents in ploidy level may result from genetic or cytoplasmic incompatibilities that are expressed either in f e r t i l i z a t i o n or at any developmental stage of the zygote between early cleavage divisions and maturity. In interspecific crosses, pollen tubes sometimes burst in the styles of foreign species (Bucholz and Blakeslee, 1929). In addition, the thick pollen tubes of polyploid species sometimes have d i f f i c u l t y growing in the slender styles of diploids or lower polyploids (Allard, 1960). These may be reasons for lower 27 Table 3 Analysis of compatibility in Meconopsis based on chromosome ploidy level No. of Cross Difference in Rank in group ploidy level compatibility (1) (2) (3) M.c. X M.q. 8 3 I M.c. X M.b. 8 3 M.b. X M.c. 8 3 M.b. X M.v. 8 2 M.c. X M.h. 4 2 M.c. X M.a. 4 3 M.v. X M.a. 4 1 II M.b. X M.h. 4 2 M.b. X M.a. 4 1 M.h. X M.c. 4 2 M.h. X M.b. 4 3 M.c. X M.v. 0 1 III M.b. X M.q. 0 1 M.h. X M.a. 0 1 M.c. X M.c. 0 1 IV M.b. X M.b. 0 1 M.h. X M.h. 0 1 M.a. X M.a. 0 2 (1) abbreviations are the same as Table 1. (2) higher ploidy level of one parent minus lower ploidy level of another parent (see text for ploidy level). (3) rank based on seeds per pollination (see Figure 7). 28 compatibility of the crosses: M_;_ cambrica x quintuplinervia, M. cambrica x M^ aculeata, M. cambrica x M^_ betonicifolia and M.  horridula x betonicifolia (Figure 7). The female parents of these crosses a l l had lower ploidy level than the male parents. Overall seed set for these crosses was less than 1.8 seeds per pollination. Meconopsis quintuplinervia has been grown in the Botanical Garden from seedlings received from Britain. It has not set seeds after several years even though hand pollination has been carried out (J. MacPhail, personal communication). Although a hybrid between M^_ quintuplinervia and M^_ cambrica has been reported (Cobb, 1984), there was no seed set from the cross of M. cambrica x M^_ quintuplinervia in the present experiment. Pollen grains of this species were viable after 10-day storage because they did successfully lead to seed set of M_^  betonicifolia. The hybrid reported between >L_ quintuplinervia and M_;_ cambrica ( Cobb, 1984) might be a reciprocal cross of M^_ cambrica (4x, female) x M.  quintuplinervia (12x, male). The barrier to pollen tube growth of polyploid species in the styles of lower polyploids may be overcome by making the reciprocal cross (Allard, 1960). Although Cobb (1984) reported that this hybrid existed sixty years ago, he 29 failed to recreate i t . An early hypothesis suggested that a particular balance of chromosome sets between the major plant parts of the developing seed (maternal tissue:endospermrembryo) was necessary for normal seed development (Peloquin, 1981). Muntzing (1933) suggested that a 2:3:2 ratio of the ploidy levels of the maternal tissue:endosperm:embryo was a requisite for normal seed development. Although i t i s not clear whether the endosperm of seeds in Meconopsis i s tr i p l o i d , the balance of the chromosome sets should be obtained more easily i f both parents have the same ploidy level. In the present experiments, crosses between parents which have the same ploidy level have the highest compatibility (Figure 7). The same level of compatibility was obtained for the reciprocal crosses: M_;_ cambrica and M_;_ betonicif o l i a , and M.  cambrica and M^_ horridula, but not for the reciprocal crosses between M^_ betonicif o l i a and M_;_ horridula. Differences between reciprocal crosses are often observed when the species that are hybridized do not have the same number of chromosomes (Allard, 1960). When the species with the larger number i s female the cross i s more successful than when i t i s male. This was 30 demonstrated in wheat and some dicotyledons (Thompson, 1930). This i s also the case in the reciprocal crosses between M.  betonicifolia and I^L_ horridula. When betonicifolia (12x) was used as the female in the cross, higher seed set could be obtained than in the reciprocal one. It i s hard to explain the high compatibility of the crosses: M. betonicifolia x aculeata and villosa x M_;_ aculeata based on information about ploidy level alone. This may be attributable to the fact that vi l l o s a and M_;_ betonicifolia can readily set seeds in gardens (Evans, 1959, and personal observation). The experiments carried out in betonicifolia show that this species had high s e l f - f e r t i l i t y and intraspecific cross compatibility (treatments IV and V of Table 2). Exposed to the natural environment after emasculation, the flower could be pollinated from the pollen of other flowers with high success (treatment I of Table 2). Pollination agents would be necessary for cross-pollination in this case. The pollinator i s unlikely to be wind, based on the result of treatment II (Table 2). Of 11 flowers treated in treatment II, two set seeds. These two flowers were inspected by opening the bags to see i f there was pollen on the stigma. This may have caused self-pollination through 31 touching the flowers. Moreover,, Meconopsis betonicifolia has large, b r i l l i a n t blue flowers and i t s pollen grains are heavy and compact. Insects are assumed to be the pollinators in this situation. No apogamy in this species was observed (treatment III of Table 2). 32 Conclusions (1) A l l interspecific crosses except cambrica x M.  quintuplinervia in this study set seeds (Table 1) but the seed setting a b i l i t y was highly correlated with the difference in ploidy level between parents. This supports the hypothesis that the degree of compatibility between species differs with difference in ploidy level between parents. The successful growth and flowering of hybrids shows that i t may be possible to combine desirable characters from both parents. (2) A l l selfed plants set seeds. The four species: M.  cambrica, M. betonicifolia, M. horridula and aculeata are s e l f - f e r t i l e . This fact supports the hypothesis that species of this genus are self-compatible. (3) The hypothesis that no apogamy exists in M.  betonicifolia i s supported. (4) Insects are the likely pollinators for betonicifolia. 33 Section B. Pollen Staining and Germination Test for Six Species in Meconopsis Introduction Henderson (1965) divided the pollen grains of Meconopsis into a number of morphological types based on shape and size of pollen grains, and thickness of exine and sexine. Of these six species, the pollen grains of M^_ quintuplinervia, M. horridula and M_;_ aculeata belong to the horridula-type while those of the other three, i.e., NL_ cambrica, M. villosa and M^_ betonicifolia are allocated to cambrica-type, villosa-type and betonicifolia-type respectively. Among the six species, Meconopsis quintuplinervia flowers in April while M^ villosa flowers in June in Vancouver. Their blooming times do not overlap. An interspecific breeding program involving these species would require pollen storage to allow controlled pollinations between species that do not flower at the same period. The object of the pollen v i a b i l i t y test was to determine 34 some of factors affecting pollen staining, pollen germination and pollen storage of the six species. Lactophenol cotton blue has been reported as an effective pollen staining agent (Radford et a l . , 1974). Of the primary factors affecting pollen germination, carbohydrates in the form of sucrose or honey are most often added to germination media as an energy supply (Stanley, 1967). The optimal concentration varies with plant species (Darlington and La Cour, 1976). Boric acid usually has the most pronounced effect in stimulating pollen germination (Goddard and Matthews, 1981; L i , 1982). 0.5-2% agar was recommended for pollen germination (Goddard and Matthews, 1981; L i , 1982). Matthews and Kraus (1981) recommended pollen storage in a desiccator with a drying agent at about 4°C. The hypotheses of this experiment were: (1) boric acid i s necessary for pollen germination in Meconopsis; (2) pollen grains of different types have different requirements for germination. 35 Materials and methods Non dehisced anthers of Meconopsis cambrica, M. vi l l o s a , M.  quintuplinervia, M. betonicifolia, M. horridula and M^_ aculeata were placed in small vials. The vials were covered with three layers of cheese cloth and placed in a desiccator which contained granular anhydrous calcium chloride. The desiccator was put in a refrigerator at 4-6°C for storage. Germination tests of the stored pollen were carried out every week. For staining and germination tests on fresh pollen, the vials were maintained at 23°C (+ 2°C) until anther dehiscence. For pollen staining tests, fresh pollen was put on a slide with a drop of lactophenol cotton blue. The percentage of pollen grains stained was recorded after 3 hours. Observations of pollen staining tests were carried out at xl60 magnification using a Photomicroscope II (Carl Zeis Oberkochen/West Germany). For each species, two slides were made. The staining percentages on each slide were then estimated by counting a random sample of 100 pollen grains. To test pollen germination, an agar medium was used. The medium consisted of 5 g sucrose , 0.5% or 1% Bacto agar 36 (Difco) with or without 0.1 mg H3BO3 . Pollen tubes of M. cambrica, M. vi l l o s a , M. horridula and M_;_ aculeata burst on 0.5% agar medium. The susequent germination test was conducted on 1% agar medium for these species. For >L_ betonicifolia, only 0.5% agar was added because no germination was observed on 1% agar medium for this species. The medium was autoclaved at 121°C (15 psi) for 15 min and then poured into 100x15 mm petri dishes. After the medium sol i d i f i e d , 20x20 mm agar blocks were cut from the dishes and placed on slides. Each was then placed in a petri dish lined with f i l t e r paper that was saturated with d i s t i l l e d water. Pollen was placed on the agar blocks with a fine hair brush. The dishes were covered to prevent moisture loss and incubated at 23°C (+_ 2°C). Pollen germination was observed after 24 hours under the microscope using the same method as for pollen staining. Pollen grains with a tube length greater than their diameter were described as having germinated. Pollen staining and germination- percentages were transformed to arcsin square root before analysis of variance. The values shown in the results are values transformed back. 37 Results Pollen stainability for a l l six species tested equalled or exceeded 85% (Table 4). No significant differences were found among the six species. Figures 8 and 9 show stained pollen grains of M_j_ betonicifolia and aculeata. They belong to different pollen types: betonicifolia pollen type and horridula pollen type respectively. Pollen of Meconopsis cambrica, M. vill o s a , M. betonicifolia, M. horridula and M^_ aculeata germinated on the a r t i f i c i a l medium tested (Table 5). Figure 10 shows the pollen tube growth of M.  aculeata. 1% agar medium was suitable for pollen germination of M. cambrica, M. vil l o s a , M. horridula and M^_ aculeata while 0.5% agar medium was suitable for pollen germination of M.  betonicifolia. Pollen germination percentage decreased with storage time (Table 5). Pollen of M^ cambrica, M. horridula and |L_ aculeata germinated after one or two-week storage while that of M^ villosa and M^_ betonicifolia did not germinate even after only one-week storage. 38 No pollen germinated on sucrose medium in the absence boric acid. 39 Table 4. Pollen staining percentage in six species of Meconopsis species percentage test result M. cambrica 85 no significant M. villosa 92 differences M. quintuplinervia 93 exist among M. betonicifolia 91 the six means. M. horridula 94 M. aculeata 91 40 Fig. 8. Stained pollen grains of Meconopsis aculeata (with one unstained pollen grain), (xl60). 41 Fig. 9. Stained pollen grains of Meconopsis betonicifolia (xl60). 42 Table 5 Pollen germination percentage in six species of Meconopsis storage time (week) 0 1 2 3 species M. cambrica 29.5 a* 7.7 b 2.5 c 0 d M. villosa 7.9 a 0 b M. quintuplinervia 0 M. betonicifolia 20.0 a 0 b M. horridula 18.5 a 13.0 b 2.9 c 0 d M. aculeata 24.0 a 11.0 b 2.9 c 0 d =germination tests were not carried out where no value i s presented. * values not labeled by the same letter in the same row differ s i g n i f i -cantly at 5% level based on Duncan's Multiple Range Test. Note: pollen of M. betonicifolia germinated on 0.5% agar medium. Pollen of other species germinated on 1% agar medium. 43 Fig. 10. Pollen tube growth of Meconopsis aculeata (xl60). kk Discussion Lactophenol cotton blue i s effective in pollen staining of the six species of Meconopsis. Pollen staining effectiveness was similar for different pollen types. Pollen of Meconopsis quintuplinervia, M. horridula and M.  aculeata belongs to the same horridula pollen type. Pollen of M.  horridula and M^_ aculeata germinated after two week storage but that of M. quintuplinervia did not. When pollen germination tests were carried out, no fresh pollen of this species was available because this species flowered earliest of the six species. It i s not known whether or not fresh pollen of >L_ quintuplinervia can germinate on the medium tested. However, this test shows that pollen of the same pollen type from different species behaves differently. Pollen from different pollen types has different requirement for germination. Pollen of M. betonicifolia (betonicifolia pollen type) germinated on the 0.5% agar medium but not on the 1% agar medium (Figures 11 and 12). One reason for this may be due to the water potential difference between 1% agar medium and the pollen grains. Pollen grains of M^ betonicifolia may have a higher 45 Fig. 11. Fresh pollen germination of Meconopsis  betonicifolia on 0.5% agar medium (xl60, 3 hour incubation). 46 12. Fresh pollen of Meconopsis betonicifolia 1% agar medium (xl60, 24 hour incubation) 47 water potential than those of the other species from the other pollen types tested, and thus the speed of hydration, exudation and germination of pollen i s reduced. Another reason may be the absence of apertures on i t s pollen grains (Figure 9). Henderson (1965) described the absence of apertures as one of the most striking features of betonicifolia pollen type. The sequence recorded by Watanabe (1955) as typifying a successful pollination includes hydration, exudation, and germination phases. Watanabe (1955, 1961) established that exudation i s an essential prelude to germination in the grasses; grains that do not exude, do not germinate. Liquid i s exuded both from the aperture and the non-apertural exine (Heslop-Harrison, 1980). More exudation was observed in 0.5% agar medium (Figure 11) than in 1% agar medium (Figure 12). It is possible that non-aperture pollen grains of M^_ betonicifolia cannot exude and thus germinate as quickly as that of the other species tested on 1% agar medium. Pollen can hydrate, exude and germinate more readily on the semi-solidified agar medium (0.5%) than on 1% agar medium because of more rapid hydration and exudation. The more fully hydrated the pollen grains, the shorter the interval before germination because less time i s needed for water uptake (Heslop-48 Harrison, 1980). Figure 12 shows that some pollen tube tips emerged but did not grow on 1% agar medium. It was assumed during the experiment that after longer incubation, these grains might produce germ tubes. Although the germination test lasted for one week for this test, no germination was observed because of bacterial and fungal contamination. This phenomenon was also observed for pollen germination of the other species tested after two-day incubation on the a r t i f i c i a l media. Contaminants can reduce germination (Goddard and Matthews, 1981). Thus, 0.5% agar medium i s more suitable than 1% agar medium for pollen germination of M_j_ betonicif o l i a . Pollen germination often needs boron as a stimulus (Li, 1982). It is also necessary for pollen germination in Meconopsis. In sucrose medium in the absence of boric acid, no pollen germination was observed. The data in Tables 4 and 5 indicate that pollen germination percentage on the a r t i f i c i a l medium was consistently lower than percentage pollen grains effectively stained by lactophenol cotton blue. This is also the case in avocado pollen (Sahar and Spiegel-Roy, 1984) and rose pollen (Pearson, 1984). Visser et a l . (1977) called stainable pollen grains "normal pollen", but 49 considered them as having only the potential to germinate. Erlanson (1931) reported that many apparently morphologically perfect grains were unable to effect f e r t i l i z a t i o n . Pollen germination alone i s not a complete measure of f e r t i l i t y since there are other contributing factors. In addition, germination of pollen in a r t i f i c i a l culture i s not the same as on the stigma. However, ±n vitro pollen germination capacity can estimate an important aspect of f e r t i l i t y (Goldy and Lyrene, 1983). 50 Conclusions (1) Fresh pollen grains of the six species in Meconopsis can be stained using lactophenol cotton blue. The stainability for the six species was 85% or more. (2) Pollen of Meconopsis cambrica, M. v i l l o s a , M. horridula and VL_ aculeata germinated on the 1% agar medium with sucrose (5 g l--'-) and boric acid (0.1 mg l --'-). Pollen of M_;_ betonicifolia germinated on the same medium with only 0.5% agar. The hypothesis that pollen from different pollen types have different requirements for pollen germination i s supported. (3) The hypothesis that boric acid i s necessary for pollen germination of the species in Meconopsis i s supported. No pollen germinated on sucrose medium in the absence of boric acid. (4) Pollen germination percent decreased with storage time. 51 Section C. Influence of Temperature and Day-length on Flowering of Meconopsis betonicifolia Introduction No previous information concerning control of flowering of Meconopsis was available. Experimental change of flowering time could be an alternative method to pollen storage for species which do not flower simultaneously. Meconopsis betonicifolia was chosen to study flowering time alteration in the growth chambers because of the availability of plant materials and their horticultural usefulness. The growth and flowering requirements for M_^_ betonicifolia were investigated. The outdoor daily mean temperature in 1983 was approximately 11°C (data from the Climatology Station of the University of British Columbia). The flowering period of M.  betonicifolia in the Botanical Garden is from early May to the end of June. The photoperiod outdoors is approximately 15-16 hours during this period in Vancouver. It i s therefore possible that long daylength is needed for i t s flowering. 52 The geographical range of betonicifolia i s in South-eastern Tibet, north-western Yunnan and northern Upper Burma at altitudes from 3,000-4,000 m (Taylor, 1934). Thus i t was assumed that plants of betonicifolia could grow and flower at low temperatures. The hypothesis for this experiment was that flowering of M.  betonicifolia i s determined by day-degree accumulation. 53 Materials and methods One-year-old potted seedlings of VL_ betonicifolia from the Botanical Garden Nursery of the University of British Columbia were studied for their flowering response in 4 daylength/temperature regimes: short days (SD, 8 hour) and long days (LD, 16 hour) combined with low temperature (LT, 6°+2°C) and high temperature (HT, 17°+2°C). Temperatures were constant day and night. The experiments were initiated on December 20, 1983 in growth chambers (Conviron Model EF7 with eight F48T12/CW/H0 fluorescent tubes and four 40 w incandescent lamps) at an energy fluence rate of 126 micro Einsteins m-2 s--*-. After 70 days, seedlings in SD, LT were transferred to LD, LT conditions because no growth was observed in SD, LT after 70 days. There were 5 seedlings for each treatment. Plants were examined every other day, and the following dates were recorded: when noticeable growth commenced (new leaves visible), when the terminal flower bud could be seen macroscopically, when the f i r s t flower unfolded and when the last flower wilted. Selfing was carried out at both low and high temperatures. 54 Results At low temperature, seedlings began to grow and the apical flower bud became visible later (Table 6). Plants needed longer from the beginning of growth to flowering at low temperature (Table 6). No difference was observed in the number of days needed to begin flowering at high temperature between LD and SD (Table 6). 55 Table 6 Daylengh and temperature change in relation to  growth and flowering of M. betonicifolia (days from the begining of experiment) treatment days to days to days to days to days from flowering period begin flower f i r s t last growth bud flower flower (1) (2) (3) (4) growth to f i r s t days flower (5) (6) dates (1984) (7) ( 3 ) - ( l ) (4)-(3) 6°C,LD 77.0 a* 157.3 a 200.3 a 17°C,LD 6.0 b 51.3 b 82.0 b 17°C,SD 6.0 b 64.0 b 93.0 b probability p<0.01 p<0.01 p<0.01 238.5 a 123.3 38.2 July 8-Aug.l5 118.5 b 76.0 36.5 Mar.12-Apr.18 110.0 b 87.0 17.0 Mar.23-Apr.9 p<0.05 values labeled by different letter in the same column differ significantly (based on Duncan's Multiple Range Test). 56 Discussion Potted seedlings of betonicifolia grew and flowered at both low temperature (6°C) and high temperature (17°C) (Figures 13 and 14). Temperature affected growth and flowering of M.  betonicifolia. High temperature induced earlier growth and flowering. The number of days needed to begin growth (new leaves visible) at low temperature i s nearly thirteen times as many as that needed at high temperature (Table 6). The number of days needed to reach flowering doubles at low temperature (Table 6). Welander (1984) reported that time until the f i r s t flower opened, decreased with increasing temperature in Aeschynanthus  speciosus. Low temperature also delays induction of flowering in Chrysanthemum morifolium (Van Ruiten and De Jing, 1984). Fitter and Hay (1981) concluded that the length of time required for completion of the annual growth cycle increases as the climate becomes cooler. The primary effect of low temperature is the reduction of rate of growth and metabolic processes. Under the same day-length (16 hour photoperiod), flowering could be induced under both low and high temperature but low temperature induced later flowering (Table 6). Accumulated 57 Fig. 13. Flowering of Meconopsis betonicifolia at low temperature (6°C, LD). 58 Fig. 14. Flowering of Meconopsis betonicifolia at high temperature (17°C) (comparison of flowering plants of M^_ betonicifolia with SD plant on the lef t and LD plant on the right). 59 temperature plays a role on flower i n i t i a t i o n within a certain range of temperature. Table 6 shows that under LD conditions, days needed to f i r s t flower at 6°C and 17°C are 200 and 82 respectively. The heat units (0°C base) were 1,200 and 1,394 Celsius degree-days respectively. The outdoor heat units (data from the University of British Columbia Climatology Station) from January to May, 1984 (flowering time of this species in the University of British Columbia Botanical Garden) were 1,220 Celsius degree-days. These values are similar numerically. It i s possible that a specific number of heat units are required to in i t i a t e flowering of this species. L i (1983) also reported that 450-570 Celsius degree-days were required for i n i t i a t i o n of plum fruit buds. However, the data collected from the Climatology Station (the University of British Columbia) may not be the same as those in the University of British Columbia Botanical Garden because of microclimate differences. Moreover, variation between growth chambers existed. Sometimes accidental temperature fluctuation within the growth chamber during the experiment occurred. These factors prevented a precise experiment from being conducted. The relationship of flower induction to accumulated heat units could 60 be tested by further experimentation. Day-length was also found to affect the growth and flowering of this species. After 70 days of low temperature treatment, some seedlings began to grow under LD but not under SD. When re-potting seedlings, i t was found that new roots had grown from seedlings subjected to LD while no new roots grew in SD (see Figures 15 and 16 for comparison). The longer day-length and greater irradiation energy stimulated growth at low temperature. Light provides energy for the synthesis of carbon compounds. Carbohydrate accumulation i s associated with light. Therefore many growth processes such as flowering, fruiting and seed production are indirectly affected by light. Root growth i s also affected, and occurs after the photosynthate requirements of the top portions of the plant have been met (Janick et a l . , 1981). Net photosynthesis is measured by both gross photosynthesis in the light and respiration in the dark. In the present experiment, long day-lengths, and thus more irradiation energy and carbohydrate accumulation, f u l f i l the photosynthate requirements of the top portions of the plant and promote both shoot and root growth. Carbohydrates cannot accumulate sufficiently to satisfy plant requirements in short days, or at least cannot accumulate 61 Fig. 15. Potted seedlings of Meconopsis betonicifolia after 70-day SD treatment at 6°C (show no growth of new roots). 62 Fig. 16. Potted seedlings of Meconopsis betonicifolia after 70-day LD treatment at 6°C (show growth of new roots). 63 as rapidly as in the long day-lengths. Day-length does not seem to have an effect on growth and flowering at high temperature as stated in the hypothesis because both LD and SD could induce growth, flower bud formation and flowering in approximately the same time (Table 6). But qualitative differences existed (Figure 14). Under SD, flowers were much smaller (5 cm in diameter). Some flower buds wilted before unfolding. The leaf area of the largest leaf was only 24 cm^. Under LD, flower size doubled. The leaf area of the largest leaf was 78 cm^. It can be seen from column 6 of Table 6 that the number of days from f i r s t flower to last flower (flowering period) under LD conditions at high temperature i s double that under SD conditions. Selfing in the growth chamber set seeds at both low and high temperature but the seed number (7-15 seeds per capsule) was only half as many as selfing of M. betonicifolia grown outdoors. The information provided by this experiment could be useful in a breeding program related to this species. The flowering time was from March 12 to April 18 at high temperature and LD in the growth chamber, was from early May to the end of June outdoors in the University of British Columbia Botanical Garden, and was from 64 July 8 to August 15 at low temperature combined with LD in the growth chamber. Pollen of betonicifolia can be used to pollinate the species which flower during these five months without pollen storage. Pollen from other species might be transferred to M.  betonicifolia to set hybrid seeds during this period of time. 65 Conclusions (1) Higher temperature (17°C) induced earlier growth and flowering than lower temperature (6°C). Temperature can change flowering time of betonicifolia under the growth chamber conditions. This i s supportive of the hypothesis that flowering of M. betonicifolia i s determined by day-degree accumulation. (2) Long daylength (16 hour photoperiod) produced more vigorous plants with larger flowers than short daylength. 66 Section D. Iii Vitro Establishment of Meconopsis betonicifolia Introduction In vitro propagation of genera from many plant families has been studied but no previous information about tissue culture of Meconopsis has been reported. Papaver, which i s closely related to Meconopsis, has been grown in tissue culture systems. The information from this genus was used for reference in the in vitro culture of Meconopsis based on the assumption that plants with close phylogenetic relationships may have similar requirements for growth and development. To establish in vitro cultures, explants, media, plant growth regulators and incubation conditions should be considered. To obtain explants, seed surface sterilization and capsule st e r i l i z a t i o n methods have been used in cotyledon and ovule culture of Papaver somniferum (Maheshwari, 1958; Ilahi, 1982). Seedlings were raised on sterilized a r t i f i c i a l medium from mature seeds and young ovules. Callus was induced from cotyledons 67 (Ilahi, 1982), hypocotyls (Schuchmann and Wellmann, 1983; Yoshikawa and Furuya, 1983), and seedling roots (Furuya et a l . , 1972). Plants of Papaver somniferum were regenerated from callus culture and flowerd in vitro (Yoshikawa and Furuya, 1983). Murashige and Skoog medium (1962) (MS medium), with some modifications, i s the one used most frequently and with greatest success (Hu and Wang, 1983). Full strength and half strength MS media were used for cotyledon culture (Ilahi, 1982), seedling root culture (Furuya et a l . , 1972) and hypocotyl culture (Yoshikawa and Furuya, 1983) of Papaver somniferum. One third strength MS also supported meristem cultures of Solanum tuberosum (Wang, 1977; Wang and Hu, 1982) and nodal segment cultures of Brassica campestris (Kuo and Tsay, 1977). The preliminary seed germination test conducted in the present study showed that one third strength MS i s suitable for seed germination of M.  betonicifolia. Plant growth regulator concentrations in the culture medium are c r i t i c a l to the control of growth and morphogenesis (Skoog and Miller, 1957). Three cytokinins are frequently used: kinetin (Kn), N 6 -benzyladenine (BA) and N6-(2-isopentenyl)-adenine (2ip). Although a particular cytokinin may not be effective for one 68 species, i t may be effective in others (Hu and Wang, 1983). 2,4-Dichlorophenoxyacetic acid (2,4-D) i s the most potent auxin in tissue culture (Hu and Wang, 1983). In tissue culture of Papaver somniferum, different concentrations of different growth regulators were used. MS medium with 2,4-D (1 mg 1 -1), Kn (0.1 mg and 7% coconut milk induced callus from hypocotyl (Yoshikawa and Furuya, 1983). Regeneration occurred in the medium supplemented with 0.1-1.0 mg 1-1 Kn (Yoshikawa and Furuya, 1983). Indole-3-acetic acid (IAA), 2,4-D, Kn and BA were used in cotyledon culture. Combination of Kn and 2,4-D at concentrations (mg I--*-): 0.1, 0.5, 1.0 and 2.0 proved to be beneficial for callus induction and i t s further proliferation (Ilahi, 1982). Kn (0.4 and 0.5 mg l " 1 ) , IAA (5 mg 1~1) and 1-naphthaleneacetic acid (NAA, 0.1 mg l--*-) were used in ovule culture (Maheshwari et a l . , 1961; Pontovich et a l . , 1966). These growth regulators enhanced proliferation of the embryonal cells and induced formation of embryos. 2,4-D (0.1 mg l - ^ ) , IAA (1 mg l " 1 ) and Kn (0.1 mg l " 1 ) were used for callus formation in seedling root culture (Furuya et a l . , 1972). Solanaceous species have been used as model systems of in  vitro studies. Among solanaceous species, i f the cytokinin:auxin 69 ratio i s <1, callus formation i s enhanced; i f >1, shoot formation is obtained (Flick et a l . , 1983). In bud culture of Chinese cabbage, Kuo and Tsay (1977) demonstrated that an exogenous cytokinin:auxin ratio >1 was necessary for good shoot growth and <1 for root differentiation. The incubation conditions were illumination of about 4,000 lux for 16 hour photoperiod at 16-18°C for shoot formation and regeneration of Papaver somniferum (Yoshikawa and Furuya, 1983). These conditions also support plant growth of Mj_ betonicifolia in the growth chambers (Section C). The aim of the present work was to determine an optimal explant and medium which supported the In vitro growth and differentiation of M_^  betonicifolia. Growth and differentiation responses ' were investigated by using different explants cultured in various media. These media contained different strengths of MS salts and different concentrations of plant growth regulators. It i s generally agreed that the lowest concentration of growth regulators which w i l l give desired morphogenetic effect should be used (Murashige, 1974). The hypothesis of this experiment was that i f cytokinin:auxin ratio i s greater than 1, then shoot 70 oliferation in M. betonicifolia would occur. 71 Materials and Methods In this experiment, Murashige and Skoog (1962) medium was used as the basic medium. Commercial MS Plant Salt Mix (Flow laboratories, cat. no. 26-330-22, lot 26330016) was supplemented with sucrose (30 g 1 -1), myo-inositol (100 mg l --*-), nicotinic acid (0.5 mg 1~1), pyridoxine HC1 (0.5 mg l - ! ) , thiamine HC1 (0.1 mg 1~1), glycine (2 mg I -!) and casein hydrolysate (1 g l --*-). The medium was adjusted to pH 5.7+0.1 with 1 N HC1 or 1 N NaOH before agar addition. The medium was dispensed into 25x150 mm culture tubes (20 ml/tube) and then autoclaved at 121°C (15 psi) for 15 minutes. A l l cultures and subcultures were incubated in growth chambers (either Sherer Model Cel 255-6 or Conviron Model EF7 with eight F48T12/CW/H0 fluorescent tubes and four 25 w or 40 w incandescent lamps) at 16-18°C, relative humidity of 65-70% and light energy of 126 micro Einsteins m-^ for 16 hour photoperiod. Explant preparation Unopened capsules of >L_ betonicifolia collected in the 72 University of British Columbia Botanical Garden were washed with d i s t i l l e d water and then rinsed in 70% ethanol for 10 sec. They were shaken in 20% "Sunbrite" bleach (1.05% sodium hypochlorite) for 5 minutes and then rinsed several times with sterilized d i s t i l l e d water before being s p l i t . A sterilized scalpel and tweezers were used to s p l i t the capsules and the seeds inside were sown on the 1/3 strength MS medium so l i d i f i e d with 6 g 1-1 Bacto agar (Difco). Seeds were incubated in the same conditions as for cultures except that some were incubated in the dark to obtain suitable hypocotyls as explants (seedlings from seeds germinated under light have no hypocotyls). In the following text, seedlings from seeds germinated in the dark are referred to as dark-raised seedlings and those from seeds germinated under light are called light-raised seedlings. After seed germination, derooted seedlings, hypocotyls and seedling roots were used as explants for in vitro establishment. To test storage conditions for seedlings, some tubes with germinated seeds were put in a cold room (4-6°C) for three months. 73 Experiment I_ To develop a medium that would support the growth of derooted seedlings of betonicifolia, several media were tested using different strengths of MS salts, cytokinins and auxin concentrations. In the f i r s t culture, the treatments were: f u l l strength MS or 1/3 strength MS sol i d i f i e d with 6 g 1 _ 1 Bacto agar. The growth regulators used were: 0 or 0.2 mg 1~1 2,4-D, 1 mg l - - * -BA, Kn or 2ip. The experiment was a 2x2x3 factorial with 45 seedlings per treatment. Shoots which survived were counted after four and eight weeks of incubation. Surviving shoots from the f i r s t culture were subcultured (subculture 1) into 1/2 strength MS medium sol i d i f i e d with 8 g agar which was used for a l l subsequent cultures because 1/3 strength MS medium gave higher survival percentage than f u l l strength MS medium but yellowish leaves in 1/3 strength MS medium suggested insufficient mineral nutrient supply. In the subcultures, the aim was to develop a medium that would stimulate multiple shoots and multiple meristems to increase propagation rate. 2 or 5 mg 1-1 BA, Kn or 2ip were used with 0.2 mg I--'- 2,4-D. The differentiated multiple meristems and multiple shoots (Figures 17-21) were counted after four and eight weeks of subculture. 74 Fig. 17. In vitro shoots from derooted seedlings of Meconopsis betonicifolia. 75 F i g . 18. In v i t r o shoots from derooted seedlings of Meconopsis b e t o n i c i f o l i a (showing c a l l u s at base of shoot) 76 Fig. 19. M u l t i p l e meristems d i f f e r e n t i a t e d from derooted s e e d l i n g of Meconopsis b e t o n i c i f o l i a . 77 Fig. 20. M u l t i p l e meristems d i f f e r e n t i a t e d from c a l l u s induced from derooted seedlings of Meconopsis b e t o n i c i f o l i a . 78 Fig. 21. Multiple shoots differentiated from derooted seedlings of Meconopsis betonicifolia. 79 The second subculture: multiple meristems and multiple shoots from the f i r s t subculture were divided and subcultured on the same medium as for the f i r s t subculture. The medium was supplemented with 0.2 or 0.5 mg l - - * - 2,4-D combined with 2 or 5 mg 1~1 2ip. After eight weeks, the surviving explants were subcultured on a medium with 0.2 mg l - - * - 2,4-D, 5 mg 1~1 2ip and 1 mg 1~1 BA to allow shoot proliferation. Experiment II The aim of this experiment was to define the ranges of concentrations of 2,4-D and 2ip that were effective in controlling morphogenetic responses of explants. Three morphological types of explants were used in this experiment: (1) hypocotyls and seedling roots from dark-raised seedlings; (2) derooted seedlings and seedling roots from light-raised seedlings; (3) petioles from light-raised seedlings. Four levels of 2ip (0, 0.1, 1.0, 10.0 mg l " 1 ) and five levels of 2,4-D (0, 0.1, 1.0, 5.0, 10.0 mg 1 _ 1) were used in a factorial experiment. There were 8 replicates for each treatment. After eight weeks, the morphological changes of different explants from each treatment were recorded. 80 S t a t i s t i c a l analysis The data for this experiment are of binomial distribution because an observation can be called either a success or a failure. A chi-square test may be used for binomial populations while the analysis of variance deals with normal populations. However, a chi-square test requires large samples to make the hypothetical frequencies equal to or larger than 5 in each c e l l . This was not always the case in the current experiments due to differences in survival between treatments. L i (1964) demonstrated that the chi-square test i s equivalent to the analysis of variance with the total mean square being used as the error term in the binomial population, with the F value being designated as F'. The analysis of variance of the current data i s only an approximate test in some cases because of disproportional and unequal sample sizes in the factorial experiment. In the text, the results are those of eight-week-old cultures or subcultures. Survival percent means percentage of surviving explants out of explants cultured. Differentiation percent means percentage of explants which differentiated into multiple meristems and multiple shoots out of surviving explants in cultures and subcultures. 81 Results Explant preparation Seedlings from seeds germinated in the dark have long hypocotyls and yellowish, smaller cotyledons while those from seeds germinated under light have no hypocotyls and have green, large cotyledons. Germinated seedlings stored at 4-6°C for three months grew well after being transferred into culture conditions. Experiment I_ In the f i r s t culture, no significant differences existed in explant survival between presence or absence of 2,4-D, or among three different cytokinins (Tables 7-9). Survival was higher in 1/3 MS than that in f u l l MS (Table 9). Of 540 derooted seedlings tested, 11 differentiated multiple shoots and multiple meristems. In the f i r s t subculture, BA, Kn or 2ip and their concentrations had no effects on differentiation percent (Tables 10 and 11). In the second subculture, concentration of 2ip had no effect on the differentiation percent but concentration of 2,4-D changed 82 differentiation percentage (Tables 12-14). The cytokinin contained in the f i r s t culture and in subculture 1 exhibited an effect on the differentiation percentage of subculture 2 (Table 14). Explants originally cultured on media containing BA and Kn developed more multiple meristems and multiple shoots than those originally cultured in 2ip containing media (this i s the same cytokinin used in the second subculture). Proliferation was obtained on the medium which contained 0.2 mg I - 1 2,4-D, 5 mg l - 1 2ip and 1 mg 1 _ 1 BA. Experiment II Where petiole explants were used, no growth or differentiation was observed. After eight weeks a l l explants died. The responses of derooted seedlings and seedling roots from light raised seedlings to exogenous auxin and cytokinin are shown in Figure 22. Figure 22 shows that when 2,4-D was 0.1 mg 1-1 or less, shoots grew well and rooted i f 2ip was 1 mg 1~~1 or less (treatments 1-3 and 5-7 of Figure 22). When 2ip concentration increased to 10 mg l - - ' - while 2,4-D concentration remained 0.1 mg or less, less than half of the shoots grew, and no rooting 83 Table 7 Survival percentage of derooted seedlings of M. betonicifolia  after treatment with 2,4-D, cytokinin and strength of MS medium (the f i r s t culture) A B C (cytokinins, 1 mg l--*-) 2,4-D strength •  (mg I" 1) of MS BA Kn 2ip 0 f u l l 20.0 20.0 28.9 1/3 44.4 48.9 51.1 0.2 f u l l 28.9 17.8 22.2 1/3 44.4 57.8 53.3 Note: 45 replicates were used for each treatment. 84 Table 8 Analysis of variance of Table _7 (the f i r s t culture) source degrees sum of mean F' probability of freedom squares squares (treatment) (11) (10.9981) (0.9998) (4.3067) (p<0.01) 2,4-D 1 0.0463 0.0463 0.1994 p>0..05 cytokinin 2 0.1815 0.0907 0.3909 p>0.05 strength of MS 1 9.8685 9.8685 42.5083 p<0.01 interactions 7 0.9019 0.1288 0.5550 p>0.05 error 528 144.1333 0.2162 total 539 125.1315 0.2322 85 Table 9 Summary of test results from Tables 1_ and £3 (the f i r s t culture) factors levels and test results 2,4-D (mg I" 1) 0 0.2 A survival (%) 35.6 a* 37.4 a strength of MS f u l l 1/3 B survival (%) 23.0 b 50.0 c cytokinin (mg 1 1) BA Kn 2ip C survival (%) 34.4 d 36.1 d 38.9 d values not labeled by the same letter in the same row differ significantly at 1% level (based on Table 8). 86 Table 10 Differentiation percentage  with different concentrations of BA, Kn or 2ip (subculture 1) concentration of cytokinins cytokinins (mg 1~1) BA Kn 2ip mean 2 27.8 11.1 23.3 20.0 b* 5 16.7 27.6 25.9 23.8 b mean 21.4 a 19.6 a 24.6 a * values labeled by the same letter in the same row or the same column do not differ (based on Table 11). Note: 18 to 30 replicates were used for each treatment. 87 Table 11 Analysis of variance of Table 10 (subculture 1) source degrees of sum of mean F' p r o b a b i l i t y freedom squares squares (treatment) (5) (0.5859) (0.1172) (0.6798) (p>0.05) concentration 1 0.0544 0.0544 0.3155 p>0.05 cytokinin 2 0.0698 0.0349 0.2024 p>0.05 in t e r a c t i o n 2 0.4616 0.2308 1.3387 p>0.05 error 149 25.9561 0.1742 t o t a l 154 26.5419 0.1724 88 Table 12 Differentiation percentage with different 2ip and 2,4-D concentration  after subculture from BA, Kn or 2ip media (subculture 2) A B C (original cytokinins) 2ip (mg I" 1) 2,4-D (mg l " 1 ) BA Kn 2ip 2 0.2 66.7 85.7 41.7 0.5 100.0 37.5 9.1 5 0.2 100.0 77.8 25.0 0.5 50.0 85.7 20.0 Note: 3 to 12 replicates were used for each treatment. 89 Table 13 Analysis of variance of Table 12 (subculture 2) source degrees of sum of mean F' probability freedom squares squares (treatment) (11) (8.0098) (0.7282) (2.8794) (p<0.01) 2ip concentration 1 0.2334 0.2334 0.9229 p>0.05 2,4-D concentration 1 1.0362 1.0362 4.0979 p<0.05 original cytokinin (2) (5.3991) (2.6995) (10.6754) (p<0.01) BA, Kn/2ip 1 5.2755 5.2755 20 .8600 p<0.01 BA/Kn 1 0.1236 0.1236 0.4887 p>0.05 interaction 7 1.3412 0.1916 0.7576 p>0.05 error 75 13.7373 0.1832 total 86 21.7471 0.2529 90 Table 14 Summary of test results from Tables 12 and 13 (subculture 2) factors levels of factors and test results 2ip (mg l " 1 ) 2 5 A differentiation (%) 45.5 a" 55.8 a 2,4-D (mg l " 1 ) 0.2 0.5 B differentiation (%) 61.4 b 39.5 c original cytokinin BA Kn 2ip C differentiation (%) 80.0 d 71.0 d 24.4 e i:" values not labeled by the same letter in the same row differ significantly at 5% level (based on Table 13). 91 or callus growth occurred (treatments 4 and 8). With increased concentration of 2,4-D (1 mg 1-1 or above), shoot growth was inhibited while callus growth increased (treatments 9-20). Shoots died and seedling roots produced callus in treatments 9, 10, 13, 14, 18 and 19.. Less than one quarter of the shoots grew, but some root and shoot base callus formed in treatments 11, 12, 15, 16 and 20. Shoots died and root callus browned in 10 mg 1~1 of 2,4-D (treatment 17). Hypocotyl and seedling root responses from dark raised seedlings to exogenous auxin and cytokinin are shown in Figure 23. This shows that callus was not induced in the absence of 2,4-D (treatments 1-4). Nor was callus induced with the absence of 2ip or 10 mg 2ip, combined with 0.1 mg l - - * - 2,4-D (treatments 5 and 8). 2ip is not c r i t i c a l to the formation of callus when the concentrations of 2,4-D exceeded 0.1 mg 1 -1. Callus obtained in treatments 10, 11, 15 and 16 showed no browning after eight weeks. When the callus was subcultured in media containing: 1.0 mg l " 1 2,4-D with 0.1 or 1.0 mg l " 1 2ip, or 5.0 mg l " 1 2,4-D with 1.0 or 10.0 mg 1_1 2ip, callus proliferation (callus multiplication) was obtained (Figures 24 and 25). 92 Fig. 22. Responses of derooted seedling and seedling root cultures from light-raised seedlings to 2ip and 2,4-D concentrations and the ratio of 2ip to 2,4-D by weight Concentration Response (mg l - ^ ) : ratio of 2ip:2,4-D Ratio Response: Legend: >l/2 shoots grew, root regeneration occurred <l/2 shoots grew, no root regeneration or callus <l/4 shoots grew, some root and some shoot base callused shoots died, root callus formed shoots died, root callus turned brown 93 Fig. 23. Responses of hypocotyl and seedling root cultures from dark-raised seedlings to 2ip and 2,4-D concentration and the ratio of 2ip to 2,4-D by weight Concentration Response (mg 1 1): >•— better results Legend: no callus, explants died 75-100% callused 94 Fig. 24. Callus induced from hypocotyls of Meconopsis betonicifolia. 95 Fig. 25. Callus induced from seedling roots of Meconopsis betonicifolia 96 Discussion Experiment I_ Strength of MS medium affects the survival of derooted seedlings in culture (Table 9). MS medium i s a high N, P, and K salt medium. When the salt concentration in the medium is lowered to one-half or one-third of the standard strength, improved cultures resulted in seedling nodal segment cultures of Grevillea  rosmarinifolia (Ben-Jaacov and Dax, 1981), in shoot tip cultures of Malus sylvestris (M. 7) (Werner and Boe, 1980), and in tuber sprout tip meristem cultures of Solanum tuberosum (Wang, 1977; Wang and Hu, 1982). Ma and Wang (1977) reported that azalea explants turned brown and died in f u l l MS strength medium, while with reduced salt concentrations, the shoot tips turned green and made substantial growth. Of the media tested, half strength MS supported excellent growth in the cotyledon cultures of opium poppy, Papaver  somniferum (Ilahi, 1982). Papaver i s a genus closely related to Meconopsis. The current study shows that survival of explants on one-97 third strength MS medium is twice as high as survival on f u l l strength MS medium. Salt concentration thus plays an important role in the explant survival of M_^  betonicifolia. Leaves produced on one-third MS medium were yellowish but survival percentage was higher than on f u l l strength MS medium. This suggested insufficient mineral nutrient because too low a salt concentration in the medium sometimes results in poor top growth in in_ vitro culture (Wang, 1978). Therefore, the subcultures were a l l conducted on half strength MS medium. In the f i r s t culture, only 2% of the derooted seedlings differentiated. The majority were from media which contained 0.2 mg 1~1 2,4-D. Cytokinin treatment was at 1 mg l - - * - in the f i r s t culture for a l l three cytokinins. When cytokinin concentration increased to 2 or 5 mg 1-1 in subculture 1, the differentiation percentage increased to 19-24% (Table 10). An interesting phenomenon was observed in the second subculture. When subcultured on the medium containing 2ip in subculture 2, the explants which had originally been cultured on media lacking 2ip (i.e., BA and Kn) produced more multiple meristems and multiple shoots than those from 2ip-containing medium (Table 14). This indicates that changing cytokinin during 98 the subculture process may improve growth and differentiation. This was also the case in apple culture. Werner and Boe (1980) reported that periodic substitution of 2ip for BA after about 4 months on BA-containing multiplication medium reduced or eliminated BA toxicity in cultures. This suggests that continual culture on the same cytokinin-containing medium might cause toxicity in cultures. The medium containing 0.2 mg 1_1 2,4-D induced more differentiation than the medium containing 0.5 mg 2,4-D in derooted seedling cultures (Table 14). The reasons for this could be: (1) the young shoot apex contains enough auxin for growth. This was demonstrated in the f i r s t culture and in Experiment II (Figure 22). Shoots can grow well without exogenous auxin; (2) cytokinins do not act alone. The interaction of cytokinin with other plant growth factors has been demonstrated in many ways (Hall, 1973). Jordan and Skoog (1971), in a study with coleoptile tips, showed that BA stimulated the synthesis of auxin. They also reported that, in tobacco tissue cultures which normally require exogenous cytokinins and auxin for growth, sufficient auxin for growth was synthesized in the presence of 99 high levels of cytokinin. This might be the case in the current experiment; (3) too high an auxin level causes dedifferentiation, not redifferentiation. This was demonstrated in Experiment II (Figure 22). Experiment II The qualitative response of different explants of M.  betonicifolia to 20 combinations of cytokinin 2ip and auxin 2,4-D was tested. Auxin:cytokinin interactions have been documented since Skoog and Miller's work (1957) with tobacco callus cultures. Their work showed the importance of auxin:cytokinin ratios in controlling shoot and root i n i t i a t i o n in vitro. The current experiments show that not only the ratio i t s e l f but also the amounts of cytokinin and auxin play important roles in the morphogenic response. In Figure 22, treatments 1-3 and 5-7 have a ratio (2ip: 2,4-D) which ranges from 0 to i n f i n i t y . Explants from these treatments a l l display similar morphological responses, i.e., more than half of the shoots grew and root regeneration occurred. 100 In these treatments, exogenous auxin and cytokinin in the media were either absent or at a very low concentration. Auxin i s essential for shoot tip culture and root i n i t i a t i o n . Since the young shoot apex is an active site for auxin biosynthesis, exogenous auxin i s not always needed in shoot tip culture (Kuo and Tsay, 1977; Ancora et a l . , 1981; Yang et a l . , 1981; Nemeth, 1981) and in root regeneration (Hasegawa, 1980; Meredith, 1979; Papachatzi et a l . , 1981). Although a small quantity of cytokinin may be synthesized by shoots grown in vitro (Koda and Okazawa, 1980), roots are the principal sites of cytokinin biosynthesis. It i s likely that endogenous cytokinin present in the explants supports shoot growth i n i t i a l l y . The regenerated roots may act as a new source of cytokinin before the residual hormone i s exhausted. In_ vitro establishment can be achieved without supplemental cytokinin in Solanum (Wang, 1977; Wang and Hu, 1982), Phaseolus (Kartha et a l . , 1981), and Fragaria (Kartha et a l . , 1980). With increased concentration of 2ip (10 mg l - ^ ) combined with 0 or 0.1 mg 1~1 2,4-D (treatments 4 and 8 of Figure 22), neither rooting, nor callus formation occurred. More than half of the shoots died. The ratio of 2ip:2,4-D in these two treatments 101 is 100 or more. It seems that this 2ip:2,4-D ratio i s too high for the i n i t i a t i o n of shoot, root or callus growth. It i s possible that callus induction depends on the presence of auxin. 0 or 0.1 mg 1~1 2,4-D cannot induce callus in derooted seedlings and root cultures in this species, but 1, 5 or 10 mg l -2,4-D induced callus (Figure 22). When the 2,4-D concentration was 1 mg or more, the ratio of 2ip:2,4-D affected morphological response (Figure 22, ratio responses). Ratios from 0.01 to 0.1 induced seedling root callus growth. Shoot growth did not occur at these low ratios. Ratios from 0.2 to 10 induced some root and shoot base callus, and also kept some shoots alive. The fact that a high exogenous cytokinin:auxin ratio favoured shoot growth has also been observed by other workers (Kuo and Tsay, 1977). The present experiment showed that the same ratio can give rise to different morphological responses. Ratios 1 and 10 can either support good shoot growth (treatments 6 and 7) or induce root callus (treatments 11, 12 and 20) depending on the 2,4-D level. The c r i t i c a l concentration in this case is between 0.1 and 1.0 mg 1 -1. With a 2,4-D concentration of 0 or 0.1 mg l - - * - , either good shoot growth and rooting can occur (treatments 1-3 and 5-102 7), or shoot growth can be poorer with no rooting and no callus formation (treatments 4 and 8). The c r i t i c a l concentration of 2ip in this case was between 1 and 10 mg 1~1. Media without 2ip (the ratio of 2ip:2,4-D i s 0) induced three kinds of morphological responses (treatments 5, 9, 13 and 17 of Figure 22) depending on the concentration of 2,4-D used: 0.1 mg 2,4-D stimulated shoot growth; 1 or 5 mg 1-1 2,4-D favoured root callus; and 10 mg 1~1 2,4-D induced l i t t l e growth. James and Newton (1977) carried out a similar experiment using strawberry plants. Their results showed that cytokinin ranging from 0.06-0.6 mg l - - * - , coupled with auxin concentrations ranging from 0.05-0.2 mg l - - * - , were most suitable for bud proliferation. Higher concentration of cytokinin inhibited bud proliferation. Higher concentration of auxin (2.0 mg 1~1) caused extensive callus formation. It can be seen from Figure 22: (1) when cytokinin:auxin ratio i s <1, four kinds of morphological responses occurred: a. more than half of the shoots grew and roots regenerated (treatment 5); b. less than a quarter of the shoots grew, some root and shoot base callus formed (treament 15); 103 c. shoots died, root callus formed (treatments 9, 10, 13, 14, 18, 19); d. shoots died, root callus browned (treatment 17). Of 9 treatments mentioned above, 7 treatments (b and c) favoured callus formation. The general rule that i f cytokininrauxin ratio i s <1, callus formation i s enhanced applies to M.  betonicifolia. This rule also applies to most solanaceous species (Flick et a l . , 1983). In general, this ratio (<1) i s not suitable for root differentiation in betonicifolia because only treatment 5 favours shoot growth and root regeneration. (2) when cytokinin:auxin ratio i s =1 or >1, three kinds of morphological responses occurred: a. more than half of the shoots grew and root regenerated (treatments 1, 2, 3, 6 and 7); b. less than half of the shoots grew, roots did not regenerate nor was callus induced (treatments 4 and 8); c. less than a quarter of the shoots grew, some root and shoot base callus formed (treatments 11, 12, 16 and 20). Although callus formed in some of treatments described above, a l l 11 treatments supported some shoot growth. The general rule that i f cytokinin:auxin ratio i s >1, shoot formation i s 104 obtained applies to betonicifolia. This rule also applies to both solanaceous species and Chinese cabbage (Flick et a l . , 1983; Kuo and Tsay, 1977). This experiment shows: concentrations of 2ip ranging from 0-1 mg l - - ' - , coupled with 2,4-D concentrations ranging from 0-0.1 mg 1~1, are suitable for shoot growth. Higher concentrations of 2,4-D (1 mg 1~1 or above) favours callus formation. Higher concentrations of 2ip (10 mg 1~1) are not suitable for shoot growth and root regeneration. The responses of hypocotyls and seedling roots from dark-raised seedlings to exogenous cytokinin and auxin are shown in Figure 23. It is clear that auxin is essential to induce callus. No callus formed without exogenous auxin. 0.1 mg l " 1 2,4-D only or i t s combination with 10 mg 2ip could not induce callus (treatments 5 and 8). When 2,4-D concentration was equal to or greater than 1 mg 1~1, no matter what concentrations of 2ip was used , callus formed in the culture. It was noted that treatments 10, 11, 15 and 16 induced white, fragile callus which showed no browning after eight weeks. The ratios of 2ip:2,4-D for these four treatments are from 0.1 to 2 (Figure 23). Therefore, concentrations of 2ip ranging from 0.1-10.0 mg 1 -1, coupled with 105 2,4-D concentrations ranging from 1-5 mg l - 1 , are suitable for callus growth. 106 Conclusions (1) General culture conditions, i.e., explants, basic medium, and incubation conditions, used in the tissue culture system of Papaver are suitable for the in vitro culture of M.  betonicifolia. It seems that plants with close phylogenetic relationships have similar requirements for growth and development in_ vitro. (2) Lower strength of MS (half or one third strength) i s better for the growth of M^_ betonicifolia than f u l l strength of MS. (3) Germinated seeds can be stored at 4-6°C for at least three months. (4) In general, i f the ratio i s <1, callus formation is enhanced; i f >1, shoot formation i s obtained. This supports the hypothesis that i f cytokinin:auxin ratio was >1, shoot proliferation in M. betonicifolia would occure. Concentrations of 2ip ranging from 0-1 mg , coupled with 2,4-D concentrations ranging from 0-0.1 mg 1~1, are suitable for shoot growth. Higher concentrations of 2ip (10 mg 1~1) are not suitable for shoot growth and root regeneration. Higher concentration of 2,4-D (1 mg 107 1-J- or above) favours callus formation in hypocotyl and seedling root cultures. 0.1-10 mg 1-1 2ip combined with 1-5 mg 1-1 2,4-D are suitable for callus growth. (5) 2 or 5 mg BA, Kn or 2ip combined with 0.2 mg 1_1 2,4-D induced multiple meristems and multiple shoots in the derooted seedling cultures of M_;_ betonicifolia. (6) Derooted seedlings cultured f i r s t on media containing BA or Kn differentiated more multiple meristems and multiple shoots after being transferred into media containing 2ip. Changing cytokinin during the subculture process may improve growth and development in vitro. 108 Overall Discussion L i t t l e research has been conducted on Meconopsis although the genus has been known for nearly 200 years. Records of hybrids exist in the genus but some of the original plants are no longer in cultivation. Rarely have specimens been preserved, and thus no description, or only a very incomplete one, can be found (Taylor, 1934). Most hybrids have arisen spontaneously and their parentage i s therefore often hypothetical. Difficulty in propagation and limited capacity for producing seed prevent the establishment of hybrids under cultivation (Taylor, 1934). Some d i f f i c u l t i e s exist in vegetative reproduction and seed storage in Meconopsis (Cox, 1934; Thompson, 1968). An entire stock may be lost i f adverse circumstances prevent the raising of young plants in one generation. Failures to maintain successful stocks of seedlings have led to the loss of many attractive species (Thompson, 1968). Therefore, i t i s very important to establish species of this genus under cultivation. Taylor (1934) pointed out that with the increase in the number of species under cultivation i t may be expected that attempts w i l l be made to 109 produce hybrids. Unfortunately, l i t t l e s c i e n t i f i c work has been conducted on this aspect for f i f t y years. This genus has not been widely used as a garden plant. The present studies provide information on the breeding and propagation of Meconopsis. They revealed: (1) interspecific crossing may be used to combine desirable characters from both parents. Most crosses conducted set hybrid seeds. This w i l l improve the horticultural usefulness of this genus and extend i t s range of cultivation; (2) pollen storage, pollen v i a b i l i t y tests, and experimental alteration of flowering time of the species in Meconopsis are practical. This w i l l f a c i l i t a t e desirable crosses between species which do not flower simultaneously; (3) in_ vitro propagation i s possible for M^_ betonicifolia. This may provide an alternative propagation method to ensure continued cultivation of this species. The same methods used in the tissue culture of M^_ betonicifolia could be tested on other species and hybrids which are not easily cultivated under garden conditions. Although most interspecific crosses conducted in the present study set seeds and viable F]_ hybrid plants were obtained, some 110 hybrids died in the later seedling stage before flowering. The hybrid (M. cambrica x horridula) which flowered in 1985 was s t e r i l e . The anthers were shrivelled and petals did not open normally. No seeds set after flowering. Hybrid i n v i a b i l i t y and hybrid s t e r i l i t y can be caused by single genes, by general incompatibility of the genotypes of the parents, or by disharmonies of gene recombination and deficiencies, duplications, and other alterations of chromosomal segments (Allard, 1960). Great diversity in chromosome number, morphology and flower colour of Meconopsis may explain this kind of genetic unbalance. Evolutionary development of Meconopsis has corresponded to changes of environmental conditions by changing morphologically (Chuang, 1981). Geographic isolation may lead to ecological isolation and increase differences in gene pool of species groups. Speciation may take place after mutation, chromosome aberration and selection. The fact that some species in this genus are polyploids probably indicates that their evolutionary diversification has taken place from an ancestral stock of high polyploid level. Because of reproductive isolation, the interspecific hybridization usually either cannot set seeds or 111 leads to hybrid i n v i a b i l i t y and hybrid s t e r i l i t y . Therefore, the hybrids produced in this study may be lost, like those hybrids reported before, i f they cannot be reproduced vegetatively or be made into allopolyploids. The present study may solve this problem. The tissue culture system established for >L_ betonicifolia could be used for propagation of Meconopsis hybrids. Hybrid seeds could be sown on a r t i f i c i a l medium and derooted seedlings could be used as explants for micropropagation. Germinated seedlings could be stored at low temperature i f necessary. Multiple shoots may be separated and allowed to proliferate in vitro. Hybrids could be preserved in this way for further study. A medium which can induce somatic embryogenesis from callus culture of M^_ betonicifolia may be developed. The maintenance of chromosomal and genetic integrity i s essential i f the goal of somatic embryogenesis i s clonal reproduction. The embryogenic cultures can produce large number of embryos per culture flask, many more than the multiple shoots generated via organogenesis (Ammirato, 1983). For breeding programs, new varieties could be developed quickly via haploid culture, somaclonal variation and somatic hybridization. This project on Meconopsis could improve 112 the horticultural usefulness of the genus. 113 Overall Conclusions and Summary (1) A l l interspecific crosses among the six species: Meconopsis cambrica, M. vi l l o s a , M. quintuplinervia, M.  betonicifolia, M. horridula and M^_ aculeata, except M^ cambrica x M. quintuplinervia, are compatible but the seed setting a b i l i t y differs with the difference in ploidy level between parents. The successful growth and flowering of hybrids show the possibility of combining desirable characters from both parents. (2) Meconopsis cambrica, M. betonicifolia, M. horridula and M. aculeata are s e l f - f e r t i l e . Meconopsis betonicifolia has high intraspecific cross compatibility. (3) Insects are the likely pollinators for M^_ betonicifolia and no apogamy exists in this species. (4) Fresh pollen of the six species can be stained using lactophenol cotton blue. Percent pollen stainability was 85% or more. 114 (5) Pollen of Meconopsis cambrica, M. vi l l o s a , M. horridula and M^_ aculeata germinated on the 1% agar medium with sucrose (5 g 1~1) and boric acid (0.1 mg 1~1). Pollen of M^ betonicifolia germinated on the same medium with only 0.5% agar. Pollen from different pollen types has different requirement for germination. (6) Pollen stored in a desiccator at about 4-6°C germinated in vitro but the germination percent decreased with storage time. Pollen of M^_ cambrica, M. horridula and M_;_ aculeata germinated after two-week or less storage while that of M_;_ vi l l o s a and M.  betonicifolia did not germinate even after only one-week storage. (7) Boric acid i s necessary for pollen germination of the six species of Meconopsis investigated. (8) Experimental alteration of flowering time of M.  betonicifolia can be obtained by controlling temperature and day-length in growth chambers. High temperature (17°C) induced earlier growth and flowering than low temperature (6°C). Long day-length (16 hour photoperiod) i s suitable to flowering of this 115 species. (9) Plants with close phylogenetic relationships have similar requirements for growth and development in_ vitro. General culture conditions used in the tissue culture system of Papaver, which is closely related to Meconopsis, are suitables for the in  vitro culture of M^_ betonicifolia. These culture conditions are: explant type, basic medium and incubation conditions. (10) Germinated seedlings can be stored at 4-6°C for at least three months. Stored seedlings grew well after being transferred to culture condition. (11) Changing cytokinin during subculture process may improve growth and development. Derooted seedlings cultured f i r s t on the media containing BA and Kn differentiated more multiple meristems and multiple shoots after being transferred into media containing 2ip than those i n i t i a l l y cultured on 2ip-containing medium. (12) Cytokinin:auxin ratio and their concentrations control 116 morphogenesis of betonicifolia in in_ vitro culture. In general, i f the ratio was <1, callus formation was enhanced; i f >1, shoot formation was obtained. 2 or 5 mg 1-1 BA, Kn or 2ip combined with 0.2 mg 2,4-D induced multiple meristems and multiple shoots in derooted seedling culture of betonicifolia. When 2,4-D concentration was 1 mg 1_1 or more, callus formed from both hypocotyls and seedling roots of this species. 0-1 mg 1~1 2ip with 0-0.1 mg 1_1 2,4-D were suitable for shoot growth in derooted seedling cultures. 0.1-10.0 mg 1~1 2ip with 1-5 mg 2,4-D were suitable for callus growth in hypocotyl and seedling root cultures. 117 Bibliography Allard, R.W. 1960. 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