Plant Cell Reports

Plant Cell Reports (1986) 5:77-80

© Springer-Verlag1986

Embryogenesis and plant regeneration of Medicago spp. in tissue culture P. Nagarajan, J. S. McKenzie, and P. D. Walton 1 Alberta Research Council, 11315 - 87 Avenue, Edmonton, Alberta, Canada T6G 2C2 Received July 29, 1985 / Revised version received December 9, 1985 - Communicated by F. Constabel

ABSTRACT Ten c u l t i v a r s and breeding l i n e s from two species of a l f a l f a (Medicago media and M. sativa) were screened for thelr--Tr--aE{Tity to p r o ~ c e e m - ~ o s and p l a n t l e t s from the root and hypocotyl under three d i f f e r e n t tissue culture protocols. The three protocols d i f f e r e d in basal s a l t composition, vitamins, hormones and cytokinin additions. That protocol having a high 2-4,D low c y t o k i n i n induction step gave the highest percentage of embryogenic c a l l i in some c u l t i v a r s and l i n e s . M. media c u l t i v a r s and breeding l i n e s had a hTgh percentage of embryoid formation. M. sativa c u l t i v a r s gave no embryoid formation. T w o - - M . ~ b r e e d i n g lines (Brl and L e l ) , which w e r e - - i n ~ d i a t e in the percentage of embryogenic c a l l i formed from explants, had the highest number of regenerated plants established in s o i l . The creeping rooted M. media c u l t i v a r Heinrichs produced the highest -percentage of embryogenic c a l l i from explants but most of these embryoids were abnormal and f a i l e d to grow in soil or vermiculite. Accordingly, successful regeneration is d i r e c t l y related to the q u a l i t y and quantity of the embryoids produced. Key words: A l f a l f a , Medicago sp., Tissue culture, Somatic embryogenesis, p--~et regeneration. INTRODUCTION A l f a l f a is grown extensively in the temperate and subtropical regions of the world. This outstanding forage crop enhances the yield of animal products (milk and meat) when used for livestock feed (Hanson, 1972). However, lack of disease and pest resistance and limited tolerance to severe stress reduces i t s productivity and adaptability in many regions.

variation may be an additional source of variation available to the plant breeder (Larkin and Scowcroft, 1981; Shepard, 1982). In order to achieve the capability of using tissue culture techniques as a tool for accomplishing plant breeding objectives, we need to know the regeneration response of various cultivars and breeding lines in tissue culture and whether regenerants have potential to grow successfully in soil media. The objective of this study was to determine the potential of 10 a l f a l f a cultivars and breeding lines, within two species, to produce in v i t r o somatic embryoids and to regenerate pl-aEtlets capable of establishing in s o i l . The cultivars and lines d i f f e r in their a b i l i t i e s to survive cold temperature stress (McKenzie and McLean, 1984 and 1985; Marble and Peterson, 1982) and to resist " a l f a l f a sickness" disease (Faechner and Bolton, 1978). MATERIALS AND METHODS Plant material consisted of five commercial cultivars (Heinrichs, Beaver, Peace, Lahontan and Maopa 69) and five breeding lines (Brl, Br2, Lel, Le2 and $7312). M. media cv. Heinrichs, Beaver, Peace and $7312 a r - e ~ e r y winterhardy having M. falcata in their background (McKenzie and McLean-~ ~8T~-I-985). M. sativa cv. 'Maopa 69' and Lahontan are not h a r d y T M a ~ n d Peterson, 1982). Brl, Br2, Lel and Le2 populations were derived from selections of M. media cv. 'Vernal, Beaver, Rambler and have resistance to ' a l f a l f a sickness' (Faechner and Bolton, 1978).

Traditional breeding methods have been slow and are expensive. I t has not always been possible to increase pest resistance, stress tolerance and forage y i e l d . Problems have related to a lack of abundance of v a r i a b i l i t y within existing populations of various cultivars.

Seed was sterilized for 15 minutes in a solution of 0.1% mercuric chloride and 0.1% sodium lauryl sulfate, then washed three times in s t e r i l e water. The seed was germinated in the dark at 29°C for 4 days on a 0.6% agar medium containing 0.2% sucrose. Callus induction from roots and hypocotyl explants occurred in the dark at 29°C over a 25 day period using Blaydes medium (Blaydes, 1966) with 2 mg/L of 2,4-D and 2 mg/L kinetin.

Tissue culture methodology has demonstrated potential for rapid clonal propagation of many plant types (Oglesby, 1978). In addition, tissue culture techniques have demonstrated that somaclonal

The three protocols used to induce embryo formation and plantlet conversion are described in Table 1. Protocol 1 and I I were developed by Stavarek et al (1980). They d i f f e r in the basal

1 Respectively." Biotechnology Department, Alberta Research Council, Agriculture Canada, Beaverlodge, Alberta, and University of Alberta, Edmonton, Alberta, Canada Offprint requests to: P. Nagarajan

78 Table I .

Three tissue culture protocols.

Purpose

Protocol I

Protocol II

Protocol I I I

Induction

None

None

SH + 13.2 mg/L KN + 8 g/L agar (4 days at

Embryo formation

LS* + 1 mg/L IAA + 6 mg/L KN* + SH, + 1 mg/L 2,4-D + 21.5 mg/L KN + 30 g/L sucrose + 6 g/L 20 g/L sucrose + 8 g/L agar agar pH 5.90 (35-45 days in pH 6.00 (35-45 days in continuous l i g h t ) continuous l i g h t )

Plantlet conversion

LS + 10 g/L sucrose + 6 g/L agar pH 5.90 (20 days in continuous l i g h t )

mg/L 2,4-D + 1.08 30 g/L sucrose + pH 5.95 29°C in dark)

BLAYDES + 2 g/L YE* + I00 mg/L myoinositol + 30 g/L sucrose + 8 g/L agar (or BOI2Y) pH 5.95 (35-45 days in continuous l i g h t ) BLAYDES + 10 g/L sucrose + 6 g/L agar pH 5.90 (20 days in continuous l i g h t )

SH + 10 g/L sucrose + 6 g/L agar pH = 5.90 (20 days in continuous l i g h t )

LS = Linsmaier and Skoog; SH = Schenk and Hilde brandt; KN = Kinetin; YE = yeast extract. salt composition, vitamins, hormones and cytokinin additives. In addition, these two protocols lack an induction step. Protocol I I I (Walker and Sato 1981) differs from I and I I by the inclusion of an induction step and the use of SH (Schenk and Hildebrandt, 1972) and Blaydes medium. Two replicates each containing eight c a l l i distributed equally between two petri-dishes each derived from both the root and hypocotyl of each c u l t i v a r or lines were cultured using each of the three protocols (Table I ) . Based on the embryogenic responses c a l l i were divided into four subjective groups (non-embryogenic, low, medium, and high embryogenic, see Table 3). The percentage of c a l l i producing embryoids were recorded and a factorial analys(s was used to evaluate the influence of the three protocol schedules, cultivars and explants on the percentage of embryogenic c a l l i formed.

RESULTS Table 2 is an analysis of variance for the percentage of c a l l i from the root and hypocotyl tissue of ten cultivars and lines showing embryogenesis when grown in three media. There were significant differences among media to induce embryo formation and there was a significant genotype x medium interaction. Protocol I I I gave the highest percentage of embryogenic c a l l i formation (Table 3). The c u l t i v a r "Heinrichs" had the greatest embryogenic response ( i . e . the highest percentage of embryogenic c a l l i but a medium response for embryoid formation - Table 3) to this protocol followed by Brl, Lel, "Beaver" and Le2. Though differences were observed in embryoid formation between root and hypocotyl explant, s t a t i s t i c a l analysis showed nonsignificance for explants, Genotype x Explants and Genotype x Explant x media interactions (Table 2). The percentage of embryogenic c a l l i formation ( i . e . embryogenic response) of cultivars and breeding lines in each protocol is given in Table 3. Considering the maximum number of embryoids produced by each embryogenic callus (based on the subjective c l a s s i f i c a t i o n ) , Brl and Lel (Table 3) gave the most promising results. The number of embryoids which

regenerated into plants and which established in soil are set out in Table 4. Genotypes Brl and Lel yielded the largest numbers of plants regenerated from callus derived embryoids. Table 2. Analysis of variance for percentage embryogenic c a l l i formation for two explant sources of a l f a l f a genotypes grown in 3 culture protocols. Source Replication Genotypes (G) Explant (E) Medium (M) GX E GX M EXM GX E X M Error

Degrees of Freedom 1 9 I 2 9 18 2 18 60

Mean Square 0.32552 0.05064 0.00208 0.12201 0.00961 0.02334 0.00013 0.00592 0.00807

F 40.32*** 6.27*** N.S. 15.11"** N.S. 2.89** N.S. N.S. N.S.

**Significant at P = 0.01%. ***Significant at P = 0.001 N.S. = not significant DISCUSSION Highly significant cultivar differences for embryogenesis were observed among the test materials. "Heinrichs" produced the highest proportion of embryogenic c a l l i (62%), and Moapa 69 and Lahontan the least (0%). Brown et al. (1985) also reported a similar percentage o-f--e~ryogenic response for Heinrichs. Whenthe total quantity of embryoids per callus (Table 3) was considered, Brl and Lel were the most productive breeding lines. Brl, Br2, Lel, Le2 and Beaver are related genotypes of M. media and they responded in a similar manner in-t-hese--6-~r-ials though they varied in the proportions of embryoids and plants they produced. The M. sativa cultivars "Peace", "Lahontan" and " M o a ~ a ' ~ r m e d poorly. The embryogenic response is evidently associated with genotype and media (G x M is significant at P = 0.01%). The results support

79 Table 3. Percentage of total embryogenic c a l l i produced from 16 c a l l i formed from the root and hypocotyl of 10 genotypes grown under three culture protocols. Percentage Embryogenic Calli and Calli Classification Genotype

Explant I

(for example in 'Heinrichs', Table 3) but s t a t i s t i c a l l y the differences were not significant. This is also evident in the G x E interaction (Table 2). Tabl e 4.

Culture Schedule II III Genotype

Heinrichs

Root Hypocotyl

0 25

ME

6 0

ME

37 62

ME

Brl

Root Hypocotyl

12 0

ME

0 0

NE

19 12

HE

Lel

Root Hypocotyl

0 0

NE

0 6

ME

19 12

HE

Beaver

Root Hypocotyl

0 0

NE

0 0

ME

19 12

ME

Le2

Root Hypocotyl

0 0

NE

0 6

LE

19 12

Br2

Root Hypocotyl

6 0

LE

6 0

LE

$7312

Root Hypocotyl

0 0

NE

0 0

NE

Root Hypocotyl

0 6

LE

0 0

NE

Root Hypocotyl

0 0

NE

0 0

NE

Root Hypocotyl

0 0

NE

0 0

NE

Protocol means

2.45

Peace Lahonton Maopa 69

*

Number of Plants Explant from Explant from root hypocotyl

Total

LE

Heinrichs Brl Lel Beaver Le2 Br2 $7312 Peace Lahonton Moapa

74 413 262 16 11 11 14 0 0 0

86 141 137 102 O 32 2 0 0 0

160 554 399 118 11 43 16 0 O 0

6 6

LE

TOTAL

801

500

1301

6 6

LE

0

LE

P l a n t l e t conversion

6 0

0

Plantlet conversion (the frequency of embryoids resulting in plants) seems to depend on the quality and quantity of embryoids produced on the c a l l i .

NE

0

1.20

Numbers of regenerated plants established in soil from root and hypocotyl embryogenic c a l l i .

NE

0

12.65

*Calli classification *a) Non-embryogenic (NE) no embryoids b) Low-embryogenic (LE) 2-5 embryoids (approximately) c) Medium-embryogenic (ME) 6-15 embryoids (approximately) d) High-embryogenic (HE) over 15 embryoids (approximately) the findings of Brown et al. (1983, 1985) who reported high regenera~on in cultivars containing M. falcata germplasm with creeping rooted character. Differences in embryogenic response with respect to media were highly significant. Generally speaking, protocol I l l gave the best response with "Heinrichs", Brl, Lel, Br2, Le2, Beaver and $7312 suggesting a requirement of high 2,4-D levels for embryo induction in these cultivars and lines. Low embryogenic enhancement was observed in "Peace" after high 2,4-D treatment (Protocol I l l ) as compared to no 2,4-D (Protocol I I ) . This suggests that certain a l f a l f a cultivars might have a 2,4-D independent differentiation mechanism for embryogenesis. Selection of such genotypes could offer scope for cytological analysis of plants for 2,4-D effects. Additionally, the source of explant tissues (root or hypocotyl) appeared to show differences with reference to embryoid formation

Though 'Heinrichs' produced embryoids in a higher proportion of c a l l i , many were aberrant ( i . e . : fused cotyledons, succulent or transluscent plantlets) and failed to establish in vermiculite or s o i l . This resulted in a low production of plantlets from embryoids. Brl and Lel gave a higher conversion rate to plantlets because they produced a greater number of embryoids per callus, and also, on subsequent subculturing, generated abundant secondary and t e r t i a r y embryoids. These results are similar to those of Lupotta (1983) who reported a process by which a recurrent harvest of regenerated embryoids could be obtained every 20 days by reculturing the somatic embryoids. The results suggest that: 1.

For some cultivars and lines embryogenesis was more successful in the protocol including an induction step.

2.

Source of explant may be important for some cultivars but in this investigation the results are not significant.

3.

Some cultivars (ex: Heinrichs) though highly regenerating, produced abundant abnormal embryoids. I t is suspected that cytological abnormalities may lead to the production of aberrant embryoids.

4.

Hence successful plantlet conversion is directly related to the quality and quantity of the embryoids produced from the cultivars and lines used in this study. The 1300 regenerants from this study have been transplanted into soil and are awaiting further morphological, physiological and cytological studies.

80 ACKNOWLEDGEMENTS Thanks are due to Miss Twyla Malcolm, Research Technologist for her help in carrying out t h i s i n v e s t i g a t i o n . Acknowledgements are also due to Dr. D.L. C u t h i e l l , Computing Department, Alberta Research Council for help in s t a t i s t i c a l analysis and Drs. D.F. Gerson (Head, Biotechnology, ARC) and C.P. Meintzer for t h e i r helpful suggestions. REFERENCES Blaydes, D.F. 1966. I n t e r a c t i o n of k i n e t i n and various i n h i b i t o r s in the growth of soybean tissue. Physiol. Plant. 19, 748-753. Brown, D.W.C., L.A. Frost and E.M. Koehl. 1983. The e f f e c t of germplasm source in the in v i t r o embryogenesis response of c u l t i v a t e d a"FFaaTfa~--. The Genetic Soc. Canada B u l l . 14: Abstr. No. D5. Brown, D.W.C. and Atanassov, A. 1985. Role of genetic background in somatic embryogenesis in Medicago. Plant Cell Tissue and Organ Culture 7F~;~-lTI~-122. Faechner, T.R. and J.L. Bolton. 1978. Genetics of resistance and s u s c e p t i b i l i t y in a l f a l f a to " a l f a l f a sick" s o i l . Can. J. Plant S c i . , 58, 945-952. Hanson, C.H. 1972. Ed. A l f a l f a Science and Technology. American Society of Agronomy, Madison, Wisc. Larkin, P.J. and Scowcroft, W.R. 1981. Somaclonal v a r i a t i o n - a novel source of v a r i a b i l i t y from c e l l cultures for plant improvement. Theor. Appl. Genet. 60:197-214. Lupotto, E. 1983. Propagation of an embryogenic c u l t u r e of Medicago sativa L. Z. Pflanzenphy~l~OTF.

Marble, V.L. and G. Peterson. 1982. A l f a l f a v a r i e t i e s and brands for C a l i f o r n i a s d i f f e r e n t s o i l s and climates pp. 15-38. In: 12th C a l i f o r n i a a l f a l f a symposium Fresno, C a l i f o r n i a . U. of C a l i f o r n i a . McKenzie, J.S. and G.E. McLean. 1984. A f i e l d test for assessing the winter hardiness of a l f a l f a in Northwestern Canada. Can. J. Plant Sci. 64:917924. McKenzie, J.S. and G.E. McLean. 1985. I d e n t i f y i n g winterhardy a l f a l f a (Medicago sativa) for Northwestern Canada. ~ t ~ to XV International Grassland Congress, Kyoto, Japan. In press). Oglesby, R.P. 1978. Tissue culture of ornamentals and flowers: Problems and perspectives. In: Propagation of higher plants through tissue c u l t u r e (K.WS. Hughes, R. Henke and M. Constantin, eds.) pp. 59-61. U.S. Dept. Energy, Washington, D.C. Schenk, R.U. and A.C. Hildebrandt. 1972. Medium and techniques for induction and growth of monocotyledonous and dicotyledonous plant cell cultures. Can. J. Bot. 50:199-204. Shepard, J.M. 1982. The regeneration of potato plants from leaf cell protoplasts. Sci. Am. 246:154-166. Stavarek, S.J., T.P. Croughan and D.W. Rains. 1980. Regeneration of plants from long term cultures of A l f a l f a c e l l s . Plant Sci. Lett. 19:253-261. Walker, K.A. and S.J. Sato. 1981. Morphogenesis in callus tissue of Medicago sativa: the role of ammonium ion in somat--~T~--e-m~yogenesis. Plant Cell Tissue and Organ Culture I , 109-121.

Embryogenesis and plant regeneration of Medicago spp. in tissue culture.

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