THE DEVELOPMENT OF PLANT REGENERATION SYSTEMS FOR THE GENETIC IMPROVEMENT OF WALNUT Walt Tu1ecke and Gale McGranahan ABSTRACT The techniques and capability to regenerate asexual embryos from walnut cotyledon tissue have been developed. Tissues from five Jug1ans regia cu1tivars, Jug1ans hindsii, and pterocarya sp. have been used. More than 100 asexual embryos have been selected from among the thousands produced. The sequence of procedures needed to obtain plants from these embryos have been worked out, and more than a dozen plants have been grown for 4 months in soil. These (and others) plants will be grown in the field and evaluated to determine whether they represent a clone or whether some are somac1ona1 variants. Similar procedures have been applied to 'Manregian' walnut endosperm tissues, and some asexual embryos have been obtained. No plants have yet been grown from these embryos. OBJECTIVE This project is designed to develop new plant regeneration systems which can be used for the genetic improvement of walnut. The project includes both short and long term objectives. The short term objective is to develop the methods required to produce asexual embryos (the process of inducing somatic embryogenesis) and plants from walnut tissues. The long term objectives are 1) to develop sources of novel germp1asm, with emphasis on somac1ona1 variants among the plants derived from asexual embryos; 2) to develop methods for the selection of asexual embryos with desirable characters, perhaps using biotechnica1 approaches for mass screening of cells or embryos for disease resistance. The system developed might also be used for in vitro genetic modifications in the future. This report covers the first objective and gives some details of the process of inducing asexual embryos and obtaining plants from them. More specific information will be available in a research paper which is being prepared for publication. PROCEDURES Immature open pollinated fruits from five walnut (Jug1ans regia L.) cu1tivars were collected at 1-2 week intervals over a 6-12 week period following full pistillate bloom. The cotyledons of the seeds in these fruits were dissected out under sterile conditions and pieces were cultured on a sequence of nutrient media (Table 1) to induce the formation of asexual embryos. Both early flowering ('Payne', 'Early Ehrhardt') and late flowering ('Franquette', 'Scharsch-Franquette', 'Plant Introduction 18256') cu1tivars were used to provide a long period of access to any given stage of development. In addition, various stages of endosperm tissue from several cu1tivars were excised and cultured on several media. This was done to obtain a callus culture which -11- - -- --
-- -- would be triploid, i.e. possess two sets of maternal and one set of paternal genes. Regeneration of plants from such tissues might provide new combinations of walnut characters. Several hundred asexual embryos were selected from among the thousands produced and were cultured according to a protocol that was developed to obtain plants from embryos (Table 2). Mature asexual embryos were given a cold treatment of 8-10 weeks at 2 C to overcome apical dormancy. Some of these embryos were grown to young plants in soil. In addition, asexual embryos were induced from cotyledon cultures of the 'California Black Walnut' (Jugflans hindsii) and from 'Wingnut' (Pterocarya sp.). RESULTS The optimal time for the induction of asexual embryogenesis was 6-11 weeks post-pollination and the percent of cotyledon pieces which were embryogenic ranged from 10-44 (Table 3). The somatic embryos appeared as early as 6 weeks and as late as 6 months after initital culture. The number produced per cotyledon piece varied from 0-26. When removed from the cotyledon piece and placed on basal medium, the asexual embryos continued to develop as embryos, produced additional adventive somatic embryos, hypertrophied, or formed abnormalities or callus. The developmental sequences for normal asexual embryogenesis included the globular, heart and cotyledonary stages and complete mature embryos. Additional asexual embryos were produced by a process of repetitive somatic embryogenesis in which asexual embryos developed at the root tips, cotyledons, and hypocotyls of asexual embryos, thus giving rise to presumably clonal embryogenic lines. Some of these lines were maintained for more than 15 months and plants have been derived from them. When mature asexual embryos without prior cold treatment were transferred to the light, they turned green, showed some root growth, but developed no leaves. Embryos given a short cold treatment (2-4 weeks) developed into plants,which exhibited the rosette characters of short internodes, poor l'eaf expansion and weak bud development. Asexual embryos given 8-10 weeks of cold developed into normal plants without rosette characters. More than a dozen plants have been grown for 4 months in soil. They developed into healthy vigorous plants with mature leaves, buds, bark, and an extensive root system. They resembled normal walnut seedlings. CONCLUSION Complete somatic embryo formation and derived plants have been obtained from cotyledon tissue of the Persian walnut. This represents the first report of somatic embryogenesis in this woody plant family, the Juglandaceae. The asexual embryos compare favorably in their developmental pattern with the descriptions given in the literature for normal sexual embryos. The capability of forming asexual embryos (embryonic competence) has also been observed in tissue cultures derived from the endosperm callus of Plant Introduction 18256 ('Manregian' ), but plants have not yet been obtained from these embryos. -12-
-- Work in the next 6 months will be directed toward two objectives. One is the selection of a population of asexual embryos from a single embryogenic line derived from one cotyledon, i.e., clonal material. These embryos will be put through the protocol to obtain plants which will be acclimatized and later planted in the field. At least 100 plants are needed to evaluate whether they are clonal (true to type) or whether they exhibit somaclonal variation. The second objective is to regenerate plants from the endosperm tissue cultures by somatic embryogenesis or. by organogenesis (bud formaticn, followed by rooting). In this case, the objective is different, since the regeneration of even a single plant would be significant. Chromosome counts would be needed to verify the genetic base of any plants derived from endosperm. Theoretically, they should be triploid, two sets of material chromosomes and one of paternal. This determination is not easy to do in walnut because the diploid chromosome number is 32 and endosperm would have 48. This approach, if successful, would provide unique sources of germplasm, especially for rootstocks. Additional work is needed on the regeneration of plants from cell suspensions of walnut tissues and from walnut leaf tiss~e. It would also be useful to develop screening methods that would allow the selection of asexual embryos (or cells) with desired characters. -13-
TABLE 1. Basal and inductive* media used for somatic embryogenesis in \'lalnut. Hedium Component nn mg/l NH4N03 Ca(N03)2.4H20 K2S04 MgS04.7H20 cac12.2h20 KH2P04 m-inositol Sucrose *.t-glutamine 17.7 8.3 8.9 3.0 1.0 1.9 0.55 87.6 1.7 1416.0 1968.0 1559.0 740.0 149.0 265.0 100.0 30,000.0 250.0 Zn(N03)2 MnS04.H20 CUS04 H3B03 Nal100 4 FeSO4. 7H20 Na2EDTA Thiamin. HCl Nicotinic acid Glycine *6-Benzylaminopurine *Kinetin *Indole-3-butyric acid }.1M 89.7 200.0 1.1 77.6 1.9 121. 5 135.0 5.9 8.1 26.6 4.4 9.3 0.05 17.0 33.4 0.25 4.8 0.39 33.8 45.4 2.0 1.0 2.0 1.0 2.0 0.01 * The inductive medium contains these canponents; the basal medium does not. Gelrite (Merck Co.) at 0.24% was used for solid media. The ph was adjusted to 5.7 with KOH. -14- --
TABLE 2. Procedures for obtaining somatic embryos and plants from walnut cotyledon tissue. PROCEDURE CONDITION TIME (WEEKS) Excise cotyledon Conditioning medium Basal medium Select somatic embryos Select cotyledon stage of somatic embryos Cotyledon maturation Break apical dormancy Leaf and root growth Transfer to peat plugs Hardening of plants Sterile technique Dark Dark Dark, basal medium Liquid tubes basal, roller Liquid basal, sucrose 6% Cold, 2 C, dark 16-hOllr photoperiod 1/2 strength basal Non-sterile, reduced humidity, soil 6-12, after full bloom 2-4 2-4, several transfers 2-4, repeated transfers 1-2 1-2 8-10 4-6 4 4 'IDTAL TIME 28-38 -15-
4>;:..' TABLE 3. Somatic embryogenesis fram cotyledon tissue of the Juglandaceae 1 I I-' I Optimal tline Cotyledon for induction Globular, explants fram Percent of somatic embryos cotyledonary, outcrossed seeds embryogenic (weeks after and mature Genus 2 2 Species of cultivar cotyledons pollination) somatic embryos Plants Juglans regia Franquette 44 7 + + Plant 33 11 + Introduction #18256 Scharsch- 29 7 + + Franquette Early 25 8 + Ehrhardt Payne 17 8 + + Juglans hindsii Rawlins 10 - + pterocarya sp. Wingnut 14 6 + + 1 Source plants were in the variety collection, Department of Parology, University of California, Davis, CA 95616. 2 + Indicates that this stage was frequently observed. - Indicates that this was not detennined.